Method for obtaining lipids from microbial cell compositions by enzymes and pH shock

Abstract

The present invention relates to a method for obtaining lipids from cells, the method involving lysing cells to form a lysed cell composition; optionally adding salt to the lysed cell composition; sequentially raising the pH of the lysed cell composition to 10 or above for a certain period of time in one or more cycles, and then lowering the pH of the lysed cell composition to a pH of less than 6 for a certain period of time to demulsify the cell composition; and separating lipids from the demulsified cell composition.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is an international application claiming priority to U.S. Provisional Application No. 62 / 818563, filed on March 14, 2019, and U.S. Provisional Application No. 62 / 818944, filed on March 15, 2019, the entire contents of each of these U.S. Provisional Applications are hereby incorporated by reference in their entirety. Technical Field

[0003] The present invention relates to a method for obtaining lipids from a composition comprising microbial cells by lysing the cells, sequentially raising and lowering the pH of the cells for one or more cycles, and isolating the lipids. The present invention also relates to lipids prepared by the method of the present invention. Background Art

[0004] A typical method for obtaining lipids (such as polyunsaturated fatty acids) from microbial cells involves growing microorganisms capable of producing the desired lipids in a fermenter, a pond, or a bioreactor; separating a fermentation broth containing microbial cell biomass; drying the microbial cell biomass; and separating lipids by solvent extraction. The steps in the separation may include diluting the fermentation broth with water; centrifuging the diluted fermentation broth; lysing the microbial cells; and extracting intracellular lipids from the lysed cells by adding a water-immiscible solvent (e.g., hexane) to the lipid-soluble mixture.

[0005] Another extraction method for removing lipid from microbial cells is to use mechanical force (e.g., homogenization), enzymatic treatment or chemical treatment to destroy cell walls to crack cells in fermentation broth. Lipid can be extracted from the resulting composition comprising lipid, microbial cell biomass and water using an organic solvent (e.g., isopropanol). Lipid can be mechanically separated from the composition, and alcohol must be removed from lipid and aqueous biomass waste streams. See, e.g., International Publication Nos. WO 01 / 76385 and WO 01 / 76715.

[0006] However, the industrial scale lipid production using any of the methods in the above method requires a large amount of volatile and flammable organic solvents, thereby creating dangerous operating conditions. In the extraction process, using organic solvents may also require the use of explosion-proof lipid recovery systems, thereby increasing the cost of lipid recovery. In addition, when extracting lipid from microbial cells, using organic solvents may produce organic solvent waste streams, which require complete solvent recovery systems or suitable disposal methods, which further increases the overall production cost of lipid extraction. For example, strict restrictions on volatile organic compounds (volatileorganic compound, VOC) discharges require more manpower and increase the cost of containers and other equipment.

[0007] Therefore, it is necessary to obtain a method for lipid from cells without using an organic solvent to minimize the above problems. Several methods for separating lipid from cells without using an organic solvent have been proposed. For example, U.S. Patent No. 6,750,048 discloses an aqueous washing method, whereby an emulsion is washed with an aqueous washing solution until substantially non-emulsified lipid is obtained. However, in some embodiments, this method requires multiple washing steps, and the multiple washing steps require a large amount of cost and time. U.S. Patent No. 7,431,952 discloses a method by which the cracked cells are centrifuged to remove cell wall fragments, and then extract and purify oily substances. However, this method provides a crude oily substance that requires a large amount of further purification. Summary of the invention

[0008] The present invention relates to a method for obtaining lipids from a composition containing microbial cells, the method comprising: lysing the microbial cells to form a lysed cell composition; optionally adding salt to the lysed cell composition; sequentially raising the pH of the lysed cell composition to a pH of 10 or above for a certain period of time in one or more cycles, and then lowering the pH of the lysed cell composition to a pH of 6 or below for a certain period of time to demulsify the cell composition; separating lipids from the demulsified cell composition; and recovering the lipids.

[0009] The present invention also relates to a method for obtaining lipids from a composition comprising microbial cells, the method comprising: enzymatically lysing the microbial cells at a pH of about 7 to about 9 and a temperature of about 60 to about 80° C. to form a lysed cell composition; optionally adding salt to the lysed cell composition; sequentially raising the pH of the lysed cell composition to a pH of 10 or above for a certain period of time in one or more cycles, and then lowering the pH of the lysed cell composition to a pH of 6 or below for a certain period of time to demulsify the cell composition; separating lipids from the demulsified cell composition; and recovering lipids.

[0010] The present invention also relates to a method for obtaining lipids from a composition comprising microbial cells, the method comprising: lysing the microbial cells at a pH of about 7 to about 9 and a temperature of about 60°C to about 80°C to form a lysed cell composition; heating the lysed cell composition to a temperature of about 80°C to about 90°C, and optionally adding salt to the lysed cell composition; sequentially raising the pH of the lysed cell composition to a pH of 10 or above for a certain period of time in one or more cycles, and then lowering the pH of the lysed cell composition to a pH of 6 or below for a certain period of time to demulsify the cell composition; separating lipids from the demulsified cell composition; and recovering the lipids.

[0011] The present invention also relates to a method for obtaining lipids from a composition comprising microbial cells, the method comprising: lysing microbial cells at a pH of about 7 to about 9 and a temperature of about 60°C to about 80°C to form a lysed cell composition; cycling the temperature of the lysed cell composition from about 4°C to about 90°C for one or more cycles; sequentially increasing the pH of the lysed cell composition to a pH of 10 or above for a certain period of time in one or more cycles, and then reducing the pH of the lysed cell composition to a pH of 6 or below for a certain period of time to demulsify the cell composition; separating lipids from the demulsified cell composition; and recovering lipids.

[0012] The present invention also relates to a method for obtaining lipids from a composition comprising microbial cells, the method comprising: lysing the microbial cells at a pH of about 7 to about 9 and a temperature of about 60°C to about 80°C to form a lysed cell composition; sequentially raising the pH of the lysed cell composition to a pH of 10 or above for a certain period of time in one or more cycles, then lowering the pH of the lysed cell composition to a pH of 6 or below for a certain period of time, and then cycling the temperature of the lysed cell composition from about 4°C to about 90°C for one or more cycles to demulsify the cell composition; separating lipids from the demulsified cell composition; and recovering the lipids.

[0013] The present invention also relates to any of the methods of the present invention, wherein the method further comprises cycling the temperature of the concentrated microbial cell composition from about 4°C to about 90°C for one or more cycles ("temperature shock") during the lysis and demulsification steps and before, between or after one or more pH shock cycles. In some embodiments, the temperature shock comprises adjusting the temperature from about 80-90°C to about 4-20°C and back to about 80-90°C.

[0014] In each of the foregoing embodiments, the recovered lipid optionally contains less than 5% organic solvent by weight or volume.

[0015] In each of the foregoing embodiments, less than 5% by weight or volume of organic solvent is used. In some embodiments, no organic solvent is used.

[0016] In some embodiments, increasing the pH comprises adding a base. In some embodiments, the base has a pK b It is 1 to 12.

[0017] In some embodiments, lowering the pH comprises adding an acid. In some embodiments, the acid has a pK a It is 1 to 12.

[0018] In some embodiments, the method comprises agitating the lysed cell composition by stirring, mixing, blending, shaking, vibrating, or a combination thereof.

[0019] In some embodiments, lysis comprises mechanical treatment, physical treatment, chemical treatment, enzymatic treatment, or a combination thereof. In some embodiments, the mechanical treatment is homogenization.

[0020] In some embodiments, the salt is added in an amount of 0.1% to 20% by weight of the lysed cell composition. In some embodiments, the salt is selected from the group consisting of alkali metal salts, alkaline earth metal salts, sulfates, and combinations thereof. In some embodiments, the salt is sodium chloride. In some embodiments, the salt is sodium sulfate.

[0021] In some embodiments, salt is omitted from the emulsification step.

[0022] In some embodiments, separating comprises centrifugation. In some embodiments, separating comprises centrifugation at a temperature of 30°C to 90°C.

[0023] In some embodiments, the method provides a lipid comprising at least 50% triglycerides by weight.

[0024] In some embodiments, the method provides a lipid having an anisidine value of 26 or less.

[0025] In some embodiments, the method provides lipids having a peroxide value of 5 or less.

[0026] In some embodiments, the method provides lipids having a phosphorus content of 100 ppm or less.

[0027] In some embodiments, the method provides lipids having at least 10% by weight of a desired polyunsaturated fatty acid (PUFA). In some embodiments, the method provides lipids having at least 10% by weight of docosahexaenoic acid ("docosahexaenoic acid, DHA"), at least 5% by weight of docosapentaenoic acid n-3 ("docosapentaenoic acid n-3, DPA n-3"), at least 10% by weight of eicosapentaenoic acid ("eicosapentaenoic acid, EPA"), and / or at least 10% by weight of arachidonic acid ("arachidonic acid, ARA").

[0028] In some embodiments, the cell is a microbial cell. In some embodiments, the method comprises concentrating a fermentation broth comprising the microbial cells.

[0029] In some embodiments, the method includes concentrating the lysed cell composition.

[0030] In some embodiments, the method comprises refining the lipids. In some embodiments, refining is selected from the group consisting of alkali refining, degumming, acid treatment, alkali treatment, cooling, heating, bleaching, deodorization, deacidification, and combinations thereof.

[0031] The present invention also relates to lipids obtained by any of the methods of the present invention.

[0032] In some embodiments, the lipids have an overall aroma intensity of 3 or less. In some embodiments, the lipids have an overall aroma intensity of 2 or less.

[0033] In some embodiments, the lipid comprises at least 10% by weight of a desired polyunsaturated fatty acid (PUFA). In some embodiments, the lipid comprises at least 10% by weight of docosahexaenoic acid ("DHA"), at least 5% by weight of docosapentaenoic acid n-3 ("DPA n-3"), at least 10% by weight of eicosapentaenoic acid ("EPA"), and / or at least 10% by weight of ARA.

[0034] In some embodiments, the lipid comprises at least 20% by weight eicosapentaenoic acid and less than 5% by weight each of arachidonic acid, docosapentaenoic acid n-6, oleic acid, linoleic acid, linolenic acid, eicosenoic acid, erucic acid, and stearidonic acid.

[0035] In some embodiments, the anisidine value of the lipid is 26 or less.

[0036] In some embodiments, the lipid is a crude lipid. In some embodiments, the crude lipid optionally has less than 5% organic solvent by weight or volume.

[0037] The present invention also relates to a lipid having an anisidine value of 26 or less, a peroxide value of 5 or less, a phosphorus content of 100 ppm or less, and optionally less than 5% organic solvent by weight or volume.

[0038] The present invention also relates to an extracted lipid comprising at least 70% by weight of a triglyceride fraction, wherein the docosahexaenoic acid content of the triglyceride fraction is at least 40% by weight, wherein the docosapentaenoic acid n-6 ​​content of the triglyceride fraction is at least 0.5% by weight to 6% by weight, and wherein the anisidine value of the oil is 26 or less. In some embodiments, the extracted lipid has a ratio of docosahexaenoic acid to docosapentaenoic acid n-6 ​​greater than 6:1.

[0039] In some embodiments, the extracted lipid is a crude lipid or a crude oil. In some embodiments, the crude lipid optionally has less than 5% organic solvent by weight or volume.

[0040] The present invention also relates to a method for obtaining lipids, the method comprising refining the crude lipids of the present invention. In some embodiments, refining is selected from the group consisting of alkali refining, degumming, acid treatment, alkali treatment, cooling, heating, bleaching, deodorization, deacidification, and combinations thereof.

[0041] The methods of the present invention provide specific advantages over methods known in the art by increasing the amount and / or quality of extracted lipid compositions, reducing the processing time of one or more steps in the method for obtaining lipids from a cell composition, reducing the amount of reagents required to obtain lipids from a cell composition, reducing the amount of hazardous chemicals utilized in the method and the amount of hazardous waste generated by the method, increasing the yield of RBDW (refined, bleached, deodorized, winterized) oil, reducing variability in commercial scale production, and reducing the overall monetary cost of extracting lipids. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings, which are incorporated herein and form a part of this specification, illustrate the present invention and, together with the description, further serve to explain the principles of the invention and enable those skilled in the relevant art to make and use the invention.

[0043] Figure 1 A schematic flow chart describing the method of the present invention is provided. DETAILED DESCRIPTION

[0044] Described herein is a method for obtaining lipids from a composition comprising microbial cells, the method comprising: lysing the microbial cells to form a lysed cell composition; optionally adding salt to the lysed cell composition; sequentially raising the pH of the lysed cell composition to 10 or above for a certain period of time in one or more cycles, followed by lowering the pH of the lysed cell composition to a pH of less than 6 for a certain period of time to demulsify the cell composition ("pH shock"); separating lipids from the demulsified cell composition; and recovering the lipids.

[0045] In various embodiments, the method includes:

[0046] a) heating a composition comprising microbial cells to a temperature of about 60° C. to about 80° C.;

[0047] b) adding one or more enzymes capable of destroying the cell walls of microbial cells for a time sufficient to lyse the microbial cells;

[0048] c) heating the lysed cell composition to a temperature of about 80° C. to about 90° C.;

[0049] d) adjusting the pH of the composition to a pH of about 10 to about 12 by adding a base and maintaining the pH of about 10 to about 12 for at least 1 hour;

[0050] e) adjusting the pH of the composition obtained in step (d) to a pH of about 4 to about 6 by adding an acid, and maintaining the pH of about 4 to about 6 for at least 0.5 hours;

[0051] f) optionally repeating steps (d) and (e) until the composition is sufficiently demulsified;

[0052] g) separating lipids from the demulsified lysed cell composition; and

[0053] h) Recovering lipids.

[0054] In some embodiments, in (a) and / or (b), the pH is adjusted from about 7 to about 9.

[0055] In some embodiments, the pH in (d) is maintained at 10 or above for a period of about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 50 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, or longer.

[0056] In some embodiments, the pH in (e) is maintained at 6 or below for a period of about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 50 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, or longer.

[0057] In various embodiments, (d) and (e) are repeated 1, 2, 3, 4 or 5 times.

[0058] In some embodiments, the method includes contacting the microbial cell composition or the cracked cell composition with a salt to promote demulsification of the cracked cell composition. In some embodiments, the salt is selected from the group consisting of alkali metal salts, alkaline earth metal salts, sulfates, and combinations thereof. In some embodiments, the salt is sodium chloride, potassium chloride, or sodium sulfate.

[0059] In some embodiments, the method comprises heating the lysed cell composition for 5 minutes to 10 hours, 10 minutes to 4 hours, 30 minutes to 2 hours, 30 minutes to 1 hour, 30 minutes to 1.5 hours, 1 hour to 1.5 hours, or 1 hour to 2 hours prior to adding the base.

[0060] In some embodiments, separating comprises centrifugation. In some embodiments, separating comprises centrifugation at a temperature of 50°C to 90°C.

[0061] In some embodiments, the method comprises: washing, centrifuging, evaporating, or a combination thereof, the fermentation broth comprising the cells prior to lysing. In some embodiments, the method comprises concentrating the culture broth comprising the cells. In some embodiments, the method comprises concentrating the lysed cell composition.

[0062] The present invention also relates to a method for obtaining lipids, the method comprising refining the crude lipids of the present invention. In some embodiments, refining is selected from the group consisting of alkali refining, degumming, acid treatment, alkali treatment, cooling, heating, bleaching, deodorization, deacidification, and combinations thereof.

[0063] Typically, the methods of the present invention do not utilize organic solvents to extract or otherwise separate lipids. Thus, in some embodiments, an organic solvent is not added to a fermentation broth containing microbial cells, to a cell composition, to a lysed cell composition, or to lipids in an amount or concentration sufficient to extract lipids during the methods of the present invention. As used herein, "organic solvent" refers to a solvent comprising at least one carbon atom. As used herein, "solvent" refers to an agent that is hydrophobic or lipophilic and is not a lipid. As used herein, "hydrophobic" refers to an agent that is repelled by a large amount of water. As used herein, "lipophilic" refers to an agent that dissolves lipids. Organic solvents not used in the methods of the present invention include, but are not limited to, polar solvents, non-polar solvents, solvents that are miscible with water, solvents that are immiscible with water, and combinations thereof. Non-limiting examples of organic solvents include substituted and unsubstituted C 4 -C 8 Alkyl (e.g., hexane, etc.), C 5 -C 12 Cycloalkyl, C 4 -C 12 Olefins, C 1 -C 8 Alcohol (e.g., isopropanol, etc.), C 1 -C 8 Aldehyde, C 4 -C 8 Ether, C 1 -C 8 Ester, C 6-C 12 Aryl, C 1 -C 8 Amide, C 5 -C 12 Heteroaryl, and combinations thereof.

[0064] In some embodiments, organic solvent can be added to the cell composition of microbial cell composition, cracking or the cell composition of demulsification.In such embodiments, organic solvent is added with a concentration less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1% or less than 0.05% by volume.Organic solvent as defined herein can be optionally added to the cell composition of cracking, for example, as a component of the alkali and / or salt contacted with the cell composition of cracking.However, in such embodiments, organic solvent exists with such concentration, and described concentration makes lipid not be extracted (that is, with a concentration less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1% or less than 0.05% by volume or weight).

[0065] In some embodiments, the method of the present invention does not include washing, such as washing with water, or the method reduces the number of washes of the lysed cell composition or the demulsified cell composition. "Washing" refers to the process of diluting the composition with, for example, water or a buffer and removing the water or buffer, for example, by centrifugation. Washing the cell composition can reduce the total yield of lipids obtained from the cell. In the present invention, washing can be reduced by 1, 2, 3 or more times.

[0066] definition

[0067] As used herein, "lipid" or "oil" refers to one or more fatty acids (including free fatty acids and fatty acid esters), phospholipids, triacylglycerols (i.e., triglycerides), diacylglycerides, monoacylglycerides, lysophospholipids, soaps, phospholipids, waxes, sterols and sterol esters, carotenoids, xanthophylls, hydrocarbons, and other lipids known to those of ordinary skill in the art. Lipids include polar lipids and neutral lipids.

[0068] As used herein, "polar lipids" refer to lipids that contain polar groups and are more soluble in polar solvents. Polar lipids include phospholipids. As used herein, "phospholipids" refer to lipids with phosphate groups. As used herein, "neutral lipids" refer to lipids that do not contain polar regions and are more soluble in non-polar solvents. Neutral lipids include triacylglycerol (TAG).

[0069] As used herein, the separation stage after, for example, centrifugation is based on separation according to specific gravity. After initial treatment but before demulsification, the "light phase" is mainly composed of materials with lower specific gravity, such as concentrated cell compositions (or "biomass"). The "heavy phase" is mainly composed of materials with higher specific gravity, such as aqueous phases (e.g., water, salts, sugars). For purposes of the present invention, the separated light phase may contain minimal residual aqueous materials.

[0070] Fatty acids are classified based on the length of the carbon chain and the saturation characteristics. Based on the number of carbons present in the chain, fatty acids are called short-chain, medium-chain, or long-chain fatty acids. When there are no double bonds between the carbon atoms, the fatty acids are called saturated fatty acids, and when double bonds are present between the carbon atoms, the fatty acids are called unsaturated fatty acids. When only one double bond is present, unsaturated long-chain fatty acids are monounsaturated, and when more than one double bond is present, unsaturated long-chain fatty acids are polyunsaturated.

[0071] The fatty acids present in the lipids can have 4 to 28 carbon atoms. In some embodiments, the lipids contain one or more polyunsaturated fatty acids. Polyunsaturated fatty acids (PUFAs) are classified based on the position of the first double bond from the methyl end of the fatty acid: ω-3 (n-3) fatty acids contain the first double bond on the third carbon, while ω-6 (n-6) fatty acids contain the first double bond on the sixth carbon. For example, docosahexaenoic acid ("DHA") is an ω-3 long chain polyunsaturated fatty acid (LC-PUFA) with a chain length of 22 carbons and 6 double bonds, commonly referred to as "22:6n-3". For the purposes of this application, long chain polyunsaturated fatty acids (LC-PUFAs) are defined as fatty acids with a carbon chain length of 18 or more, and preferably fatty acids with a carbon chain length of 20 or more, containing 3 or more double bonds. LC-PUFAs of the ω-6 series include, but are not limited to, dihomo-γ-linoleic acid (C20:3n-6), arachidonic acid (C20:4n-6) ("ARA"), docosatetraenoic acid or adrenic acid (C22:4n-6), and docosapentaenoic acid (C22:5n-6) ("DPA n-6"). LC-PUFAs of the ω-3 series include, but are not limited to, eicosatrienoic acid (C20:3n-3), eicosatetraenoic acid (C20:4n-3), eicosapentaenoic acid (C20:5n-3) ("EPA"), docosapentaenoic acid (C22:5n-3) ("DPA n-3"), and docosahexaenoic acid (C22:6n-3) ("DHA"). LC-PUFAs also include fatty acids having greater than 22 carbons and 4 or more double bonds, including, but not limited to, C24:6(n-3) and C28:8(n-3).

[0072] The terms "fatty acids", "polyunsaturated fatty acids" and "PUFAs" include not only free fatty acid forms, but also other forms, such as triacylglycerol (TAG) forms, phospholipid (PL) forms and other esterified forms. As used herein, the terms "ester" and "esterified" refer to the replacement of the hydrogen in the carboxylic acid group of the PUFA molecule by another substituent. Typical esters are known to those skilled in the art, and a discussion thereof is provided by Higuchi, T. et al., Pro-drugs as Novel Delivery Systems, Vol. 14, ACSSymposium Series, Bioreversible Carriers in Drug Design, Ed. Edward B. Roche, Amer. Pharma. Assoc., Pergamon Press (1987) and Protective Groups in Organic Chemistry, Ed. McOmie, Plenum Press, New York (1973), each of which is incorporated herein by reference in its entirety. Examples of common esters include methyl, ethyl, trichloroethyl, propyl, butyl, pentyl, tert-butyl, benzyl, nitrobenzyl, methoxybenzyl, and benzhydryl.

[0073] In some embodiments, the lipid comprises at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% PUFA by weight. In some embodiments, the lipid comprises at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% DHA by weight. In some embodiments, the lipid comprises less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, or less than 5% EPA by weight. In some embodiments, the lipid comprises less than 10%, less than 5%, less than 2%, less than 1%, or less than 0.5% sterol by weight. In some embodiments, the one or more PUFAs are present in lipids in one or more forms, such as triglycerides, diglycerides, monoglycerides, phospholipids, free fatty acids, esterified fatty acids, alkali metal salts of fatty acids, alkaline earth metal salts of fatty acids, and combinations thereof.

[0074] In some embodiments, the lipids separated after centrifugation in the methods of the invention comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or between 50% and 95%, 50% and 90%, 50% and 85%, 50% and 80%, 50% and 75%, 60% and 95%, 60% and 90%, 60% and 85%, 70% and 95%, 70% and 90%, 70% and 85%, 75% and 95%, 75% and 90%, or 75% and 85% triglycerides by weight.

[0075] In some embodiments, the triglyceride comprises at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% DHA by weight. In some embodiments, the triglyceride comprises at least 50%, at least 40%, at least 30%, at least 20%, at least 15%, at least 10%, or at least 5% EPA by weight.

[0076] As discussed herein, additional refining of the lipids after centrifugation can provide lipids comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or between 80% and 99.5%, 80% and 99%, 80% and 97%, 80% and 95%, 80% and 90%, 85% and 99.5%, 85% and 99%, 85% and 97%, 85% and 95%, 85% and 90%, 90% and 99.5%, 90% and 99%, 90% and 97%, 90% and 95%, 95% and 99.5%, 95% and 99%, 95% and 97%, 97% and 99.5%, or 98% to 99.5% triglycerides by weight.

[0077] As used herein, "cell" refers to a lipid-containing biological material, such as a biological material derived from a microorganism. As used herein, "microbial cell" or "microorganism" refers to organisms such as algae, bacteria, fungi, protists, and combinations thereof, such as unicellular organisms. In some embodiments, microbial cells are eukaryotic cells. Microbial cells suitable for use in the present invention include, but are not limited to, golden algae (e.g., microorganisms of the Stramenopiles kingdom); green algae; diatoms; dinoflagellates (e.g., microorganisms of the Dinophyceae order, including members of the genus Crypthecodinium, such as Crypthecodinium cohnii or C. cohnii); yeast (ascomycetes or basidiomycetes); and fungi of the genus Mucor and Mortierella, including but not limited to Mortierella alpina and Mortierellasect.schmuckeri.Microbial cells suitable for use in the present invention may also include, but are not limited to, genera found in the following groups: Stramenopiles, Hamatores, Proteromonads, Opalines, Develpayella, Diplophrys, Labrinthulids, Thraustochytrids, Biosecids, Oomycetes, Hypochytridiomycetes, Commation, Reticulosphaera, Pelagomonas, Pelagococcus, Ollicola, and a), Aureococcus, Parmales, Diatoms, Xanthophytes, Phaeophytes, Eustigmatophytes, Raphidophytes, Synurids, Axodines (including Rhizochromulinaales, Pedinellales, Dictyochales), Chrysomeridales, Sarcinochrysidales, Hydrurales, Hibberdiales, and Chromulinales.

[0078] In some embodiments, the microbial cell used in the present invention is a microorganism of the Labyrinthulomycota. In some embodiments, the microbial cell of the Labyrinthulomycota is a thraustochytrid, such as Schizochytrium or Thraustochytrium. According to the present invention, the term "thraustochytrid" refers to any member of the Thraustochytriales, including the Thraustochytriaceae, and the term "labyrinthulid" refers to any member of the Labyrinthulales, including the Labyrinthulaceae.

[0079] Members of the family Labyrinthaceae were previously considered members of the order Thraustochytriales, but in a more recent revision of the systematic classification of this group of organisms, Labyrinthaceae is now considered a member of the order Labyrinthales. Both Labyrinthales and Thraustochytriales are considered members of the phylum Dictyomycetes. Taxonomic theorists now generally place these two groups of microorganisms together with algae or algae-like protists of the Stramenopile lineage.

[0080] For the purposes of the present invention, microbial cell strains described as Thraustochytrids include the following organisms: Order: Thraustochytriales; Family: Thraustochytriaceae; Genus: Thraustochytrid (species: arudimentale, aureum, benthicola, globosum, kinnei, motivum, multirudimentale, pachyderm pachydermum, proliferum, roseum, and striatum), the genus Amoeboidea (species: amoeboidea, kerguelensis, minuta, profunda, radiata, sailens, and sandy chytrids). sarkariana), schizochytrops, visurgensis, yorkensis species and species BP-5601), Schizochytrium (species: aggregatum, limnaceum, mangrovei, minutum and octosporum), Nipponachytrium The present invention also provides a method for preparing a microbial cell that is suitable for use with a subject. The microbial cell may be selected from the group consisting of Japonochytrium (species: marinam), Aplanochytrium (species: haliotidis, Kerglund, Aplanochytrium, and stocchinoi), Althornia (species: crouchii), or Elina (species: marisalba and sinorificd). For the purposes of the present invention, species described in the genus Japonochytrium will be considered members of the genus Chytridium. In the present invention, the genera Aurantiacochytrium and Oblogospora are two additional genera encompassed by the phylum Digitomycetes. In some embodiments, the microbial cell belongs to the genus Thraustochystrium, Schizochytrium, and mixtures thereof.

[0081] Microbial cells suitable for use in the present invention include, but are not limited to, Labyrinthulids selected from the group consisting of: Order: Labyrinthales, Family: Labyrinthaceae, Genus: Labyrinthulids (Species: algeriensis, coenocystis, chattonii, macrocystis, macrocystis atlantica, macrocystis, marina, minuta, roscoffensis, valkanovii, vitellina, vitellina pacifica). pacifica), vitellina, and zopfii species), Labyrinthuloides (species: haliotidis and yorkensis species), Labyrinthomyxa (species: marinus species), Deiplophrys (species: archeri species), Pyrrhosorus (species: marinus species), Sorodiplophrys (species: stercorea species), and Chlamydomyxa (species: labyrinthuloides and montana species) (although there is no consensus on the exact taxonomic placement of Pyrrhosorus, Sorodiplophrys, and Chlamydomyxa).

[0082] Host cells of the phylum Dictyomycetes include, but are not limited to, deposited strains PTA-10212, PTA-10213, PTA-10214, PTA-10215, PTA-9695, PTA-9696, PTA-9697, PTA-9698, PTA-10208, PTA-10209, PTA-10210, PTA-10211, the microorganism deposited as SAM2179 (designated "Ukenkichytrium SAME 179" by the depositor), any species of the genus Ukenkichytrium (including the aforementioned species of the genus Ukenkichytrium, such as U visurgensis, U amoeboida, U sarkariana, U profunda, U radiata, U radiata), U. minuta, and U. minuta BP-5601), and includes Thraustochytrium striatum, Thraustochytrium aureum, Thraustochytrium roseum; and any species of the genus Thraustochytrium. Strains of the genus Thraustochytrium include, but are not limited to, Thraustochytrium species (23B) (ATCC 20891); Schneider (ATCC 24473); Goldstein (ATCC 34304); Goldstein (ATCC 28210); Japanese genus (LI) (ATCC 28207); ATCC 20890; ATCC 20892; mutant strains derived from any of the above microorganisms; and mixtures thereof. Schizochytrium includes, but is not limited to, Schizochytrium aggregatum, Schizochytrium limacinum, Schizochytrium species (S31) (ATCC 20888), Schizochytrium (S8) (ATCC 20889), Schizochytrium (LC-RM) (ATCC 18915), Schizochytrium (SR 21), deposited strain ATCC 28209, deposited Schizochytrium limacinum strain IFO 32693, mutant strains derived from any of the above microorganisms, and mixtures thereof. In some embodiments, the host cell is Schizochytrium or Chytrium. Schizochytrium can replicate by continuous bisection and by forming sporangia that eventually release zoospores. However, Chytrium can only replicate by forming sporangia that subsequently release zoospores. In some embodiments, the host cell of the present invention is a recombinant host cell.

[0083] Effective culture conditions for microbial cells of the present invention include but are not limited to effective culture medium, bioreactor, temperature, pH and oxygen conditions that allow lipid production. Effective culture medium refers to any culture medium of microbial cells that are usually cultivated. Such culture medium generally includes an aqueous culture medium with assimilable carbon source, nitrogen source and phosphate source and suitable salt, mineral substance, metal and other nutrients (such as vitamins). Microbial cells used for the present invention can be cultivated in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri dishes. In some embodiments, culture is carried out under temperature, pH and oxygen content that are suitable for recombinant cells.

[0084] In some embodiments, the microbial cells are capable of growing at salinity levels of 12 g / L or less, 5 g / L or less, or 3 g / L or less sodium chloride.

[0085] In some embodiments, the microbial cells produce lipids comprising ω-3PUFA and / or ω-6PUFA. In some embodiments, the microbial cells produce lipids comprising DHA, DPA (n-3), DPA (n-6), EPA, arachidonic acid (ARA), etc. and combinations thereof. Non-limiting examples of microorganisms producing lipids comprising PUFA are disclosed above and are also found in U.S. Patent Nos. 5,340,594, 5,340,742, and 5,583,019, each of which is incorporated herein by reference in its entirety.

[0086] In some embodiments, the microbial cells comprise at least 30% lipid by weight, at least 35% lipid by weight, at least 40% lipid by weight, at least 50% lipid by weight, at least 60% lipid by weight, at least 70% lipid by weight, or at least 80% lipid by weight. In some embodiments, the microbial cells used in the present invention are capable of producing at least 0.1 grams per liter per hour (g / L / h) of DHA, at least 0.2g / L / h of DHA, at least 0.3g / L / h of DHA, or at least 0.4g / L / h of DHA.

[0087] method

[0088] The method of the present invention comprises cracking microbial cell compositions or cell biomass to form cracked cell compositions. As used herein, the term "cell biomass" refers to a microbial cell population. As used herein, the term "cracking" refers to a process in which the cell wall and / or cell membrane of a cell are ruptured. In some embodiments, cracking comprises, for example, the following methods: mechanical treatment, chemical treatment, enzymatic treatment, physical treatment, or a combination thereof. In various embodiments, cracking is performed by adding an enzyme that can destroy the cell wall of a microbial cell.

[0089] As used herein, "mechanical treatment" includes, but is not limited to, homogenizing cells, applying ultrasound to cells, cold pressing cells, grinding cells, etc., and combinations thereof. In some embodiments, the method includes lysing cells by homogenization. In some embodiments, the method includes lysing cells using a homogenizer.

[0090] Homogenized cells may include, but are not limited to, methods utilizing French pressure cell presses, sonicators, homogenizers, ball mills, rod mills, gravel mills, bead mills, high pressure grinding rollers, vertical shaft impactors, industrial mixers, high shear mixers, paddle mixers, polytron homogenizers, etc., and combinations thereof. In some embodiments, cells are flowed through an optionally heated homogenizer. In some embodiments, suitable homogenization may include passing through a homogenizer 1 to 3 times under high pressure and / or low pressure. In some embodiments, the pressure during homogenization may be 150 bar to 1,400 bar, 150 bar to 1,200 bar, 150 bar to 900 bar, 150 bar to 300 bar, 300 bar to 1,400 bar, 300 bar to 1,200 bar, 300 bar to 900 bar, 400 bar to 800 bar, 500 bar to 700 bar, or 600 bar.

[0091] As used herein, physical treatment may include, but is not limited to, heating cells, drying cells, and the like, and combinations thereof.

[0092] Heating cells may include, but are not limited to, resistive heating, convection heating, steam heating, fluid bath heating, solar heating, focused solar heating, and the like, any of which may be performed in a tank, cisterns, test tubes, catheters, flasks, or other containment devices. In some embodiments, cells are heated in a tank that includes resistive coils in / on its walls. In some embodiments, cells are heated in a liquid bath that includes tubing passing therethrough. In some embodiments, cells are heated in a tank with jacketed heating, which is a tank that uses a heating "jacket" around the tank through which a heating fluid is circulated.

[0093] Drying cells can include, but are not limited to, exposure to airflow, exposure to heat (e.g., convection heat, heated surface, etc.), exposure to solar energy, freeze drying (lyophilization), spray drying, and combinations thereof. In some embodiments, drying comprises applying the cells to a rotating drum that is optionally heated.

[0094] As used herein, chemical treatment includes, but is not limited to, raising or lowering the pH of a cell or cell composition, contacting the cell or cell composition with a suitable chemical, and the like.

[0095] Increasing the pH of a cell or cell composition may include, but is not limited to, adding a base to the cell composition. In some embodiments, bases suitable for use in the present invention include, but are not limited to, hydroxide bases (e.g., LiOH, NaOH, KOH, Ca(OH) 2 etc., and combinations thereof), carbonate bases (e.g., Na 2 CO 3 , K 2 CO 3 MgCO 3 etc., and combinations thereof), bicarbonate bases (e.g., LiHCO 3 、NaHCO 3 , KHCO 3 The base can be in the form of a solid (e.g., crystals, particles, pellets, etc.) or a liquid (e.g., an aqueous solution, an alcohol solution, such as a hydroxide base in methanol, ethanol, propanol, etc.), and combinations thereof. In some embodiments, the pH of the cell composition is raised to a pH of 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, or 7 to 13, 7 to 12, 7 to 11, 7 to 10, 7 to 9, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, 9 to 12, 9 to 11, 9 to 10, 10 to 12, or 10 to 11.

[0096] In some embodiments, increasing the pH of the cell may include, but is not limited to, performing a chlor-alkali process. In some embodiments, a fermentation broth containing sodium chloride and a cell composition is electrolyzed, which results in the formation of sodium hydroxide. The formation of sodium hydroxide increases the pH of the cell. In some embodiments, the fermentation broth may contain calcium chloride or potassium chloride in place of or in addition to sodium chloride. Electrolysis of such a fermentation broth results in the formation of calcium hydroxide or potassium hydroxide, respectively, thereby increasing the pH of the cell.

[0097] Enzymatic lysis refers to the cell wall or cell membrane of the cell being cleaved by contacting the cell with one or more enzymes. Enzymes suitable for the present invention include, but are not limited to, beta-glucanase, xylanase, protease, mannanase, cellulase, hemicellulase, chitinase, pectinase, and combinations thereof. Non-limiting examples of proteases include serine proteases, threonine proteases, cysteine ​​proteases, aspartic proteases, metalloproteinases, glutamic acid proteases, alkaline proteases (alacase), and combinations thereof. Non-limiting examples of cellulases include sucrase, maltase, lactase, alpha-glucosidase, beta-glucosidase, amylase, lysozyme, neuraminidase, beta-galactosidase, alpha-mannosidase, glucuronidase, hyaluronidase, pullulanase, glucocerebrosidase, galactosylceramidase, acetylgalactosaminidase, fucosidase, hexosaminidase, iduronidase, maltase-glucoamylase, and combinations thereof. Non-limiting examples of chitinases include chitotriosidases. Non-limiting examples of pectinases include pectolyase, pectin lyase, polygalacturonase, and combinations thereof. In some embodiments, some enzymes are activated by heating. In some embodiments, the enzyme is selected from beta-glucanase, xylanase, cellulase, protease, pectinase, mannanase, amylase, and combinations thereof. In some embodiments, the enzyme is

[0098] As used herein, " cracked cell composition " refers to such composition, the composition comprises one or more cracked cells and lipid (from cracked cells) and optional culture fluid containing microbial cells, and the one or more cracked cells include cell debris and other cell inclusions. In some embodiments, microbial cells are contained in a fermentation broth or culture medium comprising microbial cells and water. In some embodiments, the cracked cell composition refers to a composition comprising one or more cracked cells, cell debris, lipid, the natural inclusions of the cell and the aqueous component from the culture fluid. In some embodiments, the cracked cell composition is in the form of an oil-in-water emulsion, and the emulsion comprises a mixture of a continuous aqueous phase and a dispersed lipid phase. In some embodiments, the dispersed lipid phase is present at a concentration of 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 5% to 60%, 5% to 50%, 5% to 40%, 5% to 30%, 5% to 20%, 10% to 60%, 10% to 50%, 10% to 40%, 20% to 60%, 20% to 50%, 20% to 40%, 30% to 60%, 30% to 50%, or 40% to 60% by weight of the emulsified lysed cell composition.

[0099] Although not bound by any particular theory, it is believed that the method of the present invention breaks or demulsifies the emulsified lysed cell composition, thereby allowing lipids to be separated from the lysed cell composition. As used herein, the terms "emulsion" and "emulsified" refer to a mixture of two or more immiscible phases or layers, wherein one phase or layer is dispersed in another phase or layer. As used herein, the terms "fragmentation", "demulsification", and "rupture" refer to the process of separating the immiscible phases or layers of an emulsion. For example, demulsification or breaking of an emulsified lysed cell composition refers to the process in which an emulsified lysed cell composition changes from an emulsion with one or more phases or layers to a composition with two or more phases or layers. For example, in some embodiments, the method of the present invention breaks the emulsified lysed cell composition from a single phase into two or more phases. In some embodiments, two or more phases include a lipid phase and an aqueous phase. In some embodiments, the method of the present invention breaks the emulsified lysed cell composition from one or more phases into at least three phases. In some embodiments, the three phases include a lipid phase, an aqueous phase, and a solid phase. In some embodiments, the three phases include a lipid phase, an emulsion phase, and an aqueous phase.

[0100] In some embodiments, the methods of the invention demulsify the lysed cell composition to form a demulsified cell composition by removing or disrupting at least 75% of the emulsion, at least 80% of the emulsion, at least 85% of the emulsion, at least 90% of the emulsion, at least 95% of the emulsion, at least 99% of the emulsion. In some embodiments, the methods of the invention demulsify the lysed cell composition by removing or disrupting 75% to 99% of the emulsion, 75% to 95% of the emulsion, 75% to 90% of the emulsion, 75% to 85% of the emulsion, 75% to 80% of the emulsion, 80% to 99% of the emulsion, 80% to 95% of the emulsion, 80% to 90% of the emulsion, 80% to 85% of the emulsion, 85% to 99% of the emulsion, 85% to 95% of the emulsion, 85% to 90% of the emulsion, 90% to 99% of the emulsion, 90% to 95% of the emulsion, or 95% to 99% of the emulsion by weight or volume.

[0101] In some embodiments, the cells may be washed and / or pasteurized prior to lysing the cells.

[0102] In some embodiments, washing the cells comprises using an aqueous solution, such as water, to remove any extracellular water-soluble or water-dispersible compounds. In some embodiments, the cells can be washed once, twice, three times or more. In some embodiments, pasteurizing the cells comprises heating the cells to a temperature sufficient to inactivate any unwanted enzymes or activate any desired enzymes, such as any enzymes that may degrade lipids or reduce PUFA production. In some embodiments, the pasteurization temperature is about 60° C. to about 80° C. In some embodiments, the cells can be washed first and then pasteurized.

[0103] In some embodiments, the method of the present invention includes increasing the pH of the cell composition to crack and / or demulsify the cell composition. In some embodiments, the method of the present invention includes increasing the pH of the cell composition of the cracking to demulsify the cell composition of the cracking. In some embodiments, increasing the pH includes contacting the cell composition or the cell composition of the cracking with an alkali. In some embodiments, the method of the present invention includes contacting the cell composition of the cracking with an alkali to demulsify the cell composition of the cracking. As used herein, "contacting" refers to combining a cell composition or a cell composition of the cracking with a second composition (e.g., by adding a composition to a cell composition or a cell composition of the cracking, by adding a cell composition or a cell composition of the cracking to a composition, etc.). As used herein, "composition" may include a pure material or a combination of two or more materials, substances, excipients, parts, etc. Contacting the cell composition of the cracking with the first alkali increases the pH of the cell composition of the cracking.

[0104] In some embodiments, the lysed cell composition is contacted with a first base for a certain period of time, followed by contact with a first acid for a certain period of time, followed by heating, agitation, or a combination thereof, followed by contact with a second or subsequent base, followed by contact with a second or subsequent acid to provide a demulsified composition.

[0105] In some embodiments, the pK of the base is b is 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 3 to 10, 3 to 6, 3 to 5, 4 to 10, 4 to 8, 4 to 6, 5 to 10, or 5 to 8. As used herein, the term "pK b ” refers to the association constant K of the base b The negative logarithm of K b It refers to the equilibrium constant for the dissociation of a base in water.

[0106] Suitable bases for use in the present invention include, but are not limited to, hydroxide bases (e.g., LiOH, NaOH, KOH, Ca(OH) 2 etc., and combinations thereof), carbonate bases (e.g., Na2 CO 3 , K 2 CO 3 MgCO 3 etc., and combinations thereof), bicarbonate bases (e.g., LiHCO 3 、NaHCO 3 , KHCO 3 The base may be in the form of a solid (e.g., crystals, particles, pellets, etc.) or a liquid (e.g., an aqueous solution, an alcohol solution, such as a hydroxide base in methanol, ethanol, propanol, etc.), and combinations thereof. Therefore, a solvent may optionally be present in the base used in the present invention. As used herein, "solvent" refers to a hydrophobic or lipophilic agent. As used herein, "hydrophobic" refers to an agent that is repelled by a large amount of water. As used herein, "lipophilic" refers to an agent that dissolves in lipids.

[0107] In some embodiments, contacting the cell composition or the lysed cell composition with a base increases the pH of the lysed cell composition. In some embodiments, contacting the lysed cell composition with a base increases the pH of the lysed cell composition to 9 or more, 10 or more, 11 or more, 12 or more, or about 9 to about 14, about 9 to about 13.5, about 9 to about 13, about 9 to about 12.5, about 9 to about 12, about 9 to about 11.5, about 9 to about 11, about 9 to about 10.5, about 9 to about 10, about 9 to about 9.5, about 9.5 to about 14, about 9.5 to about 13.5, about 9.5 to about 13, about 9.5 to about 12.5, about 9.5 to about 12, about 9.5 to about 11.5, about 9.5 to about 11, about 9.5 to about 10.5, about 9.5 to about 10, about 10 to about 14, about 10 to about 13.5, about 10 to about 13, about 10 to about 12.5, about 10 to about 12, about 10 to about 11.5, about 10 to about 11, about 10 to about 10.5, about 10.5 to about 14, about 10.5 to about 13.5, about 10.5 to about 13, about 10.5 to about 12.5, about 10.5 to about 12, about 10.5 to about 11.5, about 10.5 to about 11, about 11 to about 14, about 11 to about 13.5, about 11 to about 13, about 11 to about 12.5, about 11 to about 12, about 11 to about 11.5, about 11.5 to about or about 14, about 11.5 to about 13.5, about 11.5 to about 13, about 11.5 to about 12.5, about 11.5 to about 12, about 12 to about 14, about 12 to about 13.5, about 12 to about 13, about 12 to about 12.5, about 12.5 to about 14, about 12.5 to about 13.5, about 12.5 to about 13, about 13 to about 14, about 13 to about 13.5, or about 13.5 to about 14.

[0108] In some embodiments, the base is added in an amount of about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 3% to about 6%, about 4% to about 6%, about 5% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 5%, about 3% to about 4%, or about 4% to about 5% by weight (or volume) of the cell culture fluid to increase the pH.

[0109] In some embodiments, the pH is lowered by adding an acid. In some embodiments, the pK of the acid is a is 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 3 to 10, 3 to 6, 3 to 5, 4 to 10, 4 to 8, 4 to 6, 5 to 10, or 5 to 8. As used herein, the term "pK a ” refers to the acid-base association constant K a The negative logarithm of K a It refers to the equilibrium constant for the dissociation of an acid in water.

[0110] Acids include, but are not limited to, sulfuric acid; phosphoric acid; hydrochloric acid; hydrobromic acid; hydroiodic acid; hypochlorous acid; chlorous acid; chloric acid; perchloric acid; fluorosulfonic acid; nitric acid; fluoroantimonic acid; fluoroboric acid; hexafluorophosphoric acid; chromic acid; boric acid; acetic acid; citric acid; formic acid; and combinations thereof. In some embodiments, the pH is selected from about 6.5 or less; about 6 or less; about 5.5 or less; about 5 or less; about 4.5 or less; about 4 or less; about 3.5 or less; about 3 or less; about 2.5 or less; about 2 or less; about 1.5 or less; about 1 or less; and about 0.5 or less. In other embodiments, the pH is selected from about 0.5 to about 6.5; about 0.5 to about 6; about 0.5 to about 5.5; about 0.5 to about 5; about 0.5 to about 4.5; about 0.5 to about 4; about 0.5 to about 3.5; about 0.5 to about 3; about 0.5 to about 2.5; about 0.5 to about 2; about 0.5 to about 1.5; about 0.5 to about 1; about 1 to about 7; about 1 to about 6.5; about 1 to about 6; about 1 to about 5.5; about 1 to about 5; about 1 to about 4.5; about 1 to about 4; about 1 to about 3.5; about 1 to about 3; about 1 to about 2.5; about 1 to about 2; about 1 to about 1.5; about 1.5 to about 7; about 1.5 to about 6.5; about 1.5 to about 6; about 1.5 to about 5.5; about 1.5 to about 5; about 1.5 to about 4.5; about 1.5 to about 4; about 1.5 to about 3.5; about 1.5 to about 3; about 1.5 to about 2.5; about 1.5 to about 2; about 2 to about 7; about 2 to about 6.5; about 2 to about 6; about 2 to about 5.5; about 2 to about 5; about 2 to about 4.5; about 2 to about 4, about 2 to about 3.5; about 2 to about 3; about 2 to about 2.5; about 2.5 to about 7; about 2.5 to about 6.5; about 2.5 to about 6; about 2.5 to about 5.5; about 2.5 to about 5; about 2.5 to about 4.5; about 2.5 to about 4; about 2.5 to about 3.5; about 2.5 to about 3; about 3 to about 7; about 3 to about 6.5; about 3 to about 6; about 3 to about 5.5; about 3 to about 5; about 3 to about 4.5; about 3 to about 4; about 3 to about 3.5; about 3.5 to about 7; about 3.5 to about 6.5; about 3.5 to about 6; about 3.5 to about 5.5; about 3.5 to about 5; about 3.5 to about 4.5; about 3.5 to about 4; about 4 to about 7; about 4 to about 6.5; about 4 to about 6; about 4 to about 5.5; about 4 to about 5; about 4 to about 4.5; about 4.5 to about 7; about 4.5 to about 6.5; about 4.5 to about 6; about 4.5 to about 5.5; about 4.5 to about 5; about 5 to about 7; about 5 to about 6.5; about 5 to about 6; about 5 to about 6.5; about 5 to about 6; about 5 to about 5.5; about 5.5 to about 7; about 5.5 to about 6.5; about 5.5 to about 6; and about 6 to about 6.5.

[0111] In some embodiments, the acid is added in an amount of about 0.5% to about 1%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 3% to about 6%, about 4% to about 6%, about 5% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 5%, about 3% to about 4%, or about 4% to about 5% by weight (or volume) of the cell culture fluid to lower the pH.

[0112] In some embodiments, the method includes contacting the cell composition or the lysed cell composition with a salt to promote demulsification of the lysed cell composition. As used herein, "salt" refers to a demulsified composition prepared by contacting the cell composition or the lysed cell composition with a metal (e.g., an alkali metal, an alkaline earth metal, a transition metal, etc.) or a positively charged compound (e.g., NH 4 + The ionic compound formed by replacing the hydrogen ions from the acid with an ion (e.g., sodium chloride, sodium carbonate, calcium chloride, potassium sulfate, magnesium sulfate, monosodium glutamate, ammonium sulfate, potassium chloride, ferric chloride, ferric sulfate, aluminum sulfate, and a combination thereof. In some embodiments, the salt used in the present invention is selected from: sodium chloride, sodium sulfate, sodium carbonate, calcium chloride, potassium sulfate, magnesium sulfate, monosodium glutamate, ammonium sulfate, potassium chloride, ferric chloride, ferric sulfate, aluminum sulfate, and a combination thereof. In some embodiments, the salt does not include NaOH. The salt can be added in the form of a solid (e.g., crystalline, amorphous, pelleted and / or granular form) and / or in the form of a solution (e.g., dilute solution, saturated solution, or supersaturated solution) containing, for example, water, alcohol, etc., and a combination thereof.

[0113] In some embodiments, the salt is added in an amount of 5 g / l to 25 g / l, 5 g / l to 10 g / l, 10 g / l to 15 g / l, 15 g / l to 20 g / l, 20 g / l to 25 g / l, or 10 g / l to 20 g / l.

[0114] In some embodiments, when salt is added to demulsify the cell composition or the lysed cell composition, the temperature of the cell composition or the lysed cell composition is less than or equal to 60°C, less than or equal to 55°C, less than or equal to 45°C, less than or equal to 40°C, less than or equal to 35°C, less than or equal to 30°C, or less than or equal to 25°C. In some embodiments, when salt is added to demulsify the cell composition or the lysed cell composition, the temperature of the lysed cell composition is between 0°C and 60°C, 0°C and 55°C, 0°C and 50°C, 0°C and 45°C, 0°C and 40°C, 0°C and 35°C, 0°C and 30°C, 0°C and 25°C, 20°C and 60°C, 20°C and 55°C, 20°C and 50°C, 20°C and 45°C, 20°C and 40°C, 20°C and 35°C, 20°C and 30°C, 30°C and 60°C, 30°C and 55°C, 30°C and 50°C, 30°C and 45°C, 30°C and 40°C, 30°C and 40°C, 40°C and 60°C, 40°C and 55°C, 40°C and 50°C, or 50°C and 60°C.

[0115] In some embodiments, the method includes a temperature shock cycle before, during or after one or more pH shock cycles in a pH shock cycle." Temperature shock cycle" refers herein to raising a cell composition or a cracked cell composition to a temperature of about 80°C, about 85°C, about 90°C, about 95°C or higher for a certain period of time (e.g., about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 50 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours or longer) and then reducing to a temperature of about 30°C, about 25°C, about 20°C, about 15°C, about 10°C, about 5°C or about 4°C for a certain period of time (e.g., about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 50 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours or longer) to promote demulsification. In some embodiments, the temperature shock cycle occurs before the pH shock cycle. In other embodiments, a temperature shock cycle occurs between two or more of the pH shock cycles.

[0116] In some embodiments, the method comprises contacting a cell composition or a lysed cell composition with 20% or less, 15% or less, 10% or less, 7.5% or less, 5% or less, or 2% or less of salt by weight of the lysed cell composition or cell composition. In some embodiments, a method comprises contacting a cell composition or a lysed cell composition with 0.1% to 20%, 0.1% to 15%, 0.1% to 10%, 0.5% to 20%, 0.5% to 15%, 0.5% to 10%, 0.5% to 5%, 0.5% to 4%, 0.5% to 3%, 0.5% to 2.5%, 0.5% to 2%, 0.5% to 1.5%, 0.5% to 1%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 1% to 4%, %, 1% to 3%, 1% to 2.5%, 1% to 2%, 1% to 1.5%, 1.5% to 5%, 1.5% to 4%, 1.5% to 3%, 1.5% to 2.5%, 1.5% to 2%, 2% to 20%, 2% to 15%, 2% to 10%, 2% to 5%, 2% to 4%, 2% to 3%, 2% to 2.5%, 2.5% to 5%, 2.5% to 4%, 2.5% to 3%, 3% to 5%, 3% to 4%, 4% to 5%, 5% to 20%, 5% to 15%, 5% to 10%, 10% to 20%, 10% to 15%, or 15% to 20% of a salt thereof. For example, when the lysed cell composition weighs 1,000 kg, contacting with 0.5% to 20% by weight of the salt requires mixing 5 kg to 200 kg of the salt with the lysed cell composition.

[0117] In some embodiments, the method includes heating the cell composition or the cracked cell composition to break the emulsion of the cracked cell composition. In some embodiments, the cell composition or the cracked cell composition is heated for a sufficiently long period of time so that the alkali and / or salt breaks the emulsion of the cell composition or the cracked cell composition. In some embodiments, the method includes heating the cell composition or the cracked cell composition for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 66 hours, at least 72 hours, at least 78 hours, at least 84 hours, at least 90 hours or at least 96 hours. In some embodiments, the method comprises heating the lysed cell composition for 5 minutes to 96 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, 5 minutes to 1 hour, 10 minutes to 4 hours, 10 minutes to 2 hours, 10 minutes to 1 hour, 1 hour to 2 hours, 1 hour to 96 hours, 1 hour to 84 hours, 1 hour to 72 hours, 1 hour to 60 hours, 1 hour to 48 hours, 1 hour to 36 hours, 1 hour to 24 hours, 1 hour to 4 hours, 4 hours to 96 hours, 4 hours to 84 hours, 4 hours to 72 hours, 4 hours to 60 hours, 4 hours to 48 hours, 4 hours to 36 From 1 to 24 hours, from 4 hours to 24 hours, from 8 hours to 96 hours, from 8 hours to 84 hours, from 8 hours to 72 hours, from 8 hours to 60 hours, from 8 hours to 48 hours, from 8 hours to 36 hours, from 8 hours to 24 hours, from 8 hours to 12 hours, from 12 hours to 96 hours, from 12 hours to 84 hours, from 12 hours to 72 hours, from 12 hours to 60 hours, from 12 hours to 48 hours, from 12 hours to 36 hours, from 12 hours to 24 hours, from 24 hours to 96 hours, from 24 hours to 84 hours, from 24 hours to 72 hours, from 24 hours to 60 hours, from 24 hours to 48 hours, or from 24 hours to 36 hours.

[0118] In some embodiments, the cell composition or lysed cell composition can be heated at a temperature of at least 10°C, at least 20°C, at least 25°C, at least 30°C, at least 35°C, at least 40°C, at least 45°C, at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C, or at least 100°C. In some embodiments, the method comprises heating the cell composition or the lysed cell composition at a temperature of 10°C to 100°C, 10°C to 90°C, 10°C to 80°C, 10°C to 70°C, 20°C to 100°C, 20°C to 90°C, 20°C to 80°C, 20°C to 70°C, 30°C to 100°C, 30°C to 90°C, 30°C to 80°C, 30°C to 70°C, 40°C to 100°C, 40°C to 90°C, 40°C to 80°C, 50°C to 100°C, 50°C to 90°C, 50°C to 80°C, 50°C to 70°C, 60°C to 100°C, 60°C to 90°C, 60°C to 80°C, 70°C to 100°C, 70°C to 90°C, 80°C to 100°C, 80°C to 90°C, or 90°C to 100°C. In some embodiments, salts can be added to the cell composition or lysed cell composition during heating.

[0119] In some embodiments, the cell composition or the lysed cell composition can be heated in a closed system or a system with an evaporator. In some embodiments, the cell composition or the lysed cell composition can be heated in a system with an evaporator so that a portion of the water present in the cell composition or the lysed cell composition is removed by evaporation. In some embodiments, a method includes heating the cell composition or the lysed cell composition in a system with an evaporator to remove up to 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the water by weight present in the cell composition or the lysed cell composition. In some embodiments, a method comprises heating a cell composition or a lysed cell composition in a system having an evaporator to remove 1% to 50%, 1% to 45%, 1% to 40%, 1% to 35%, 1% to 30%, 1% to 25%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 5%, 5% to 50%, 5% to 45%, 5% to 40%, 5% to 35%, 5% to 30%, 5% to 25%, 5% to 20%, 5% to 15%, 5% to 10%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, From 10% to 15%, 15% to 50%, 15% to 45%, 15% to 40%, 15% to 35%, 15% to 30%, 15% to 25%, 15% to 20%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, 20% to 25%, 25% to 50%, 25% to 45%, 25% to 40%, 25% to 35%, 25% to 30%, 30% to 50%, 30% to 45%, 30% to 40%, 30% to 35%, 35% to 50%, 35% to 45%, 35% to 40%, 40% to 50%, 40% to 45%, or 45% to 50%.

[0120] In some embodiments, the method includes keeping the cell composition or the lysed cell composition in a container for a predetermined time to demulsify the lysed cell composition. In some embodiments, the method includes keeping the cell composition or the lysed cell composition in a container for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 66 hours, at least 72 hours, at least 78 hours, at least 84 hours, at least 90 hours or at least 96 hours. In some embodiments, the method comprises maintaining the cell composition or lysed cell composition at each pH level in the cycle for 5 minutes to 96 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, 5 minutes to 1 hour, 10 minutes to 4 hours, 10 minutes to 2 hours, 10 minutes to 1 hour, 1 hour to 96 hours, 1 hour to 84 hours, 1 hour to 72 hours, 1 hour to 60 hours, 1 hour to 48 hours, 1 hour to 36 hours, 1 hour to 24 hours, 1 hour to 4 hours, 1 hour to 2 hours, 4 hours to 96 hours, 4 hours to 84 hours, 4 hours to 72 hours, 4 hours to 60 hours, 4 hours to 48 hours. From 1 to 36 hours, from 4 hours to 36 hours, from 4 hours to 24 hours, from 8 hours to 96 hours, from 8 hours to 84 hours, from 8 hours to 72 hours, from 8 hours to 60 hours, from 8 hours to 48 hours, from 8 hours to 36 hours, from 8 hours to 24 hours, from 8 hours to 12 hours, from 12 hours to 96 hours, from 12 hours to 84 hours, from 12 hours to 72 hours, from 12 hours to 60 hours, from 12 hours to 48 hours, from 12 hours to 36 hours, from 12 hours to 24 hours, from 24 hours to 96 hours, from 24 hours to 84 hours, from 24 hours to 72 hours, from 24 hours to 60 hours, from 24 hours to 48 hours, or from 24 hours to 36 hours.

[0121] In some embodiments, the method comprises contacting the lysed cell emulsion with an antioxidant, optionally before and / or after pasteurization. Antioxidants suitable for use in the present invention include, but are not limited to, tocopherols, tocotrienols, polyphenols, resveratrol, flavonoids, carotenoids, lycopene, carotene, lutein, ascorbic acid, palmitic acid ascorbyl ester, and the like, and combinations thereof.

[0122] As used herein, the term "agitation" refers to the process of affecting movement in a lysed cell composition by applying a force. In some embodiments, the methods of the present invention include agitating the cell composition or the lysed cell composition by stirring, mixing, blending, oscillating, vibrating, or a combination thereof. In some embodiments, the process of agitating the cell composition or the lysed cell composition demulsifies the cell composition or the lysed cell composition.

[0123] In some embodiments, the methods of the invention comprise agitating the cell composition or the lysed cell composition at 0.1 hp / 1,000 gal lysed cell composition to 10 hp / 1,000 gal lysed cell composition, 0.5 hp / 1,000 gal lysed cell composition to 8 hp / 1,000 gal lysed cell composition, 1 hp / 1,000 gal lysed cell composition to 6 hp / 1,000 gal lysed cell composition, or 2 hp / 1,000 gal lysed cell composition to 5 hp / 1,000 gal lysed cell composition.

[0124] In some embodiments, the method of the present invention includes stirring the cell composition or the cracked cell composition using an agitator. In some embodiments, the agitator is a dispersed agitator, which disperses alkali and / or salt in the cell composition or the cracked cell composition. In some embodiments, the agitator has one or more impellers. As used herein, "impeller" refers to a device arranged to transfer motion to the cell composition or the cracked cell composition when rotating. Impellers suitable for the present invention include straight blade impellers, Rushton blade impellers, axial flow impellers, radial flow impellers, concave blade disc impellers, high-efficiency impellers, propellers, blades, turbines, etc., and combinations thereof. In some embodiments, a method comprises agitating a cell composition or a lysed cell composition using an agitator having an impeller tip speed of 90 ft / min to 1,200 ft / min, 200 ft / min to 1,000 ft / min, 300 ft / min to 800 ft / min, 400 ft / min to 700 ft / min, or 500 ft / min to 600 ft / min. In some embodiments, a method comprises agitating a cell composition or a lysed cell composition using an agitator having an impeller tip speed of 350 cm / sec to 900 cm / sec, 350 cm / sec to 850 cm / sec, 350 cm / sec to 800 cm / sec, 350 cm / sec to 750 cm / sec, 350 cm / sec to 700 cm / sec, 350 cm / sec to 650 cm / sec, 350 cm / sec to 600 cm / sec. , 350 cm / sec to 550 cm / sec, 350 cm / sec to 500 cm / sec, 350 cm / sec to 450 cm / sec, 350 cm / sec to 400 cm / sec, 400 cm / sec to 900 cm / sec, 400 cm / sec to 850 cm / sec, 400 cm / sec to 800 cm / sec, 400 cm / sec to 750 cm / sec, 400 cm / sec to 700 cm / sec, 400 cm / sec to 65 0 cm / s, 400 cm / s to 600 cm / s, 400 cm / s to 550 cm / s, 400 cm / s to 500 cm / s, 400 cm / s to 450 cm / s, 450 cm / s to 900 cm / s, 450 cm / s to 850 cm / s, 450 cm / s to 800 cm / s, 450 cm / s to 750 cm / s, 450 cm / s to 700 cm / s, 450 cm / s to 850 cm / s, 450 cm / s to 800 cm / s, 450 cm / s to 750 cm / s, 450 cm / s to 700 cm / s, 450 cm / s to 850 cm / s, 450 cm / s to 800 cm / s / sec to 650 cm / sec, 450 cm / sec to 600 cm / sec, 450 cm / sec to 550 cm / sec, 450 cm / sec to 500 cm / sec, 500 cm / sec to 900 cm / sec, 500 cm / sec to 850 cm / sec, 500 cm / sec to 800 cm / sec, 500 cm / sec to 750 cm / sec, 500 cm / sec to 700 cm / sec, 500 cm / sec to 650 cm / sec,500 cm / sec to 600 cm / sec, 500 cm / sec to 550 cm / sec, 550 cm / sec to 900 cm / sec, 550 cm / sec to 850 cm / sec, 550 cm / sec to 800 cm / sec, 550 cm / sec to 750 cm / sec, 550 cm / sec to 700 cm / sec, 550 cm / sec to 650 cm / sec, 550 cm / sec to 600 cm / sec, 600 cm / sec to 900 cm / sec, 600 cm / sec to 850 cm / sec, 600 cm / sec to 800 cm / sec, 600 cm / sec to 750 cm / sec, 600 cm / sec to 700 cm / sec, 600 cm / sec to 650 cm / sec, 650 cm / sec m / s to 900 cm / s, 650 cm / s to 850 cm / s, 650 cm / s to 800 cm / s, 650 cm / s to 750 cm / s, 650 cm / s to 700 cm / s, 700 cm / s to 900 cm / s, 700 cm / s to 850 cm / s, 700 cm / s to 800 cm / s, 700 cm / s to 750 cm / s, 750 cm / s to 900 cm / s, 750 cm / s to 850 cm / s, 750 cm / s to 800 cm / s, 800 cm / s to 900 cm / s, 800 cm / s to 850 cm / s, or 850 cm / s to 900 cm / s. As used herein, "impeller tip speed" refers to the speed of the outermost portion of the impeller as it rotates about its central axis.

[0125] In some embodiments, agitation (and optionally additional steps as described herein) is performed in a container comprising an impeller, wherein the ratio of impeller diameter to container volume is 0.1 to 0.5, 0.1 to 0.4, 0.2 to 0.5, 0.2 to 0.4, 0.3 to 0.5, or 0.3 to 0.4.

[0126] In some embodiments, agitation (and optionally additional steps as described herein) is performed in a vessel comprising an impeller wherein the ratio of the impeller diameter to the vessel inner diameter is at least 0.25, at least 0.34, at least 0.65, between 0.25 and 0.65, between 0.25 and 0.33, between 0.3 and 0.6, between 0.3 and 0.5, between 0.3 and 0.4, between 0.34 and 0.65, between 0.34 and 0.6, between 0.34 and 0.55, between 0.37 and 0.55, between 0.4 and 0.65, between 0.4 and 0.6, between 0.4 and 0.5, or between 0.42 and 0.55.

[0127] In some embodiments, agitation comprises mixing the cell composition or lysed cell composition such that the cell composition or lysed cell composition is subjected to flow conditions described by a Reynolds number of 10 to 10,000, 1,000 to 10,000, 1,500 to 10,000, or 2,000 to 10,000. In some embodiments, the Reynolds number of the lysed cell emulsion during agitation is 2,000 or more, 3,000 or more, or 5,000 or more, or 2,000 to 10,000, 3,000 to 10,000, or 5,000 to 10,000.

[0128] In some embodiments, a method comprises agitating a cell composition or a lysed cell composition for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 42 hours, at least 48 hours, at least 54 hours, at least 60 hours, at least 66 hours, at least 72 hours, at least 78 hours, at least 84 hours, at least 90 hours, or at least 96 hours. In some embodiments, a method comprises agitating a cell composition or a lysed cell composition for 5 minutes to 96 hours, 5 minutes to 4 hours, 5 minutes to 2 hours, 5 minutes to 1 hour, 10 minutes to 4 hours, 10 minutes to 2 hours, 10 minutes to 1 hour, 1 hour to 96 hours, 1 hour to 84 hours, 1 hour to 72 hours, 1 hour to 60 hours, 1 hour to 48 hours, 1 hour to 36 hours, 1 hour to 24 hours, 1 hour to 4 hours, 4 hours to 96 hours, 4 hours to 84 hours, 4 hours to 72 hours, 4 hours to 60 hours, 4 hours to 48 hours, 4 hours to 36 hours, 4 hours to From 8 hours to 24 hours, from 8 hours to 96 hours, from 8 hours to 84 hours, from 8 hours to 72 hours, from 8 hours to 60 hours, from 8 hours to 48 hours, from 8 hours to 36 hours, from 8 hours to 24 hours, from 8 hours to 12 hours, from 12 hours to 96 hours, from 12 hours to 84 hours, from 12 hours to 72 hours, from 12 hours to 60 hours, from 12 hours to 48 hours, from 12 hours to 36 hours, from 12 hours to 24 hours, from 20 hours to 40 hours, from 24 hours to 96 hours, from 24 hours to 84 hours, from 24 hours to 72 hours, from 24 hours to 60 hours, from 24 hours to 48 hours, or from 24 hours to 36 hours.

[0129] In some embodiments, a method comprises agitating and heating a cell composition or a lysed cell composition simultaneously to break the cell composition or the lysed cell composition. In some embodiments, a method comprises agitating a cell composition or a lysed cell composition at a temperature of at least 10°C, at least 20°C, at least 25°C, at least 30°C, at least 35°C, at least 40°C, at least 45°C, at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C, or at least 100°C. In some embodiments, a method comprises agitating a cell composition or a lysed cell composition at a temperature of 10°C to 100°C, 10°C to 90°C, 10°C to 80°C, 10°C to 70°C, 20°C to 100°C, 20°C to 90°C, 20°C to 80°C, 20°C to 70°C, 30°C to 100°C, 30°C to 90°C, 30°C to 80°C, 30°C to 70°C, 40°C to 100°C, 40°C to 90°C, 40°C to 80°C, 50°C to 100°C, 50°C to 90°C, 50°C to 80°C, 50°C to 70°C, 60°C to 100°C, 60°C to 90°C, 60°C to 80°C, 70°C to 100°C, 70°C to 90°C, 80°C to 1000°C, 80°C to 90°C, or 90°C to 100°C.

[0130] In some embodiments, the various combinations of forming a cracked cell composition, contacting the cracked cell composition with a first alkali or improving the pH of the cracked cell composition, contacting the cracked cell composition with a salt, heating the cracked cell composition, and stirring the cracked cell composition can be carried out in a single container. In some embodiments, the various combinations of forming a cell composition, contacting the cell composition with an alkali or improving the pH of the cell composition, contacting the cell composition with a salt, heating the cell composition, and stirring the cell composition can be carried out in a single container. In some embodiments, a single container includes a fermentation vessel. In some embodiments, the fermentation vessel can have a volume of at least 20,000 liters, at least 50,000 liters, at least 100,000 liters, at least 120,000 liters, at least 150,000 liters, at least 200,000 liters, or at least 220,000 liters. In some embodiments, the fermentation vessel can have a volume of 20,000 liters to 220,000 liters, 20,000 liters to 100,000 liters, 20,000 liters to 50,000 liters, 50,000 liters to 220,000 liters, 50,000 liters to 150,000 liters, 50,000 liters to 100,000 liters, 100,000 liters to 220,000 liters, 100,000 liters to 150,000 liters, 100,000 liters to 120,000 liters, 150,000 liters to 220,000 liters, 150,000 liters to 200,000 liters, or 200,000 liters to 220,000 liters.

[0131] In some embodiments, a certain amount of cell composition or lysed cell composition formed in a container can be transferred to one or more agitation containers. In some embodiments, the agitation container can have a volume of at least 20,000 liters, at least 30,000 liters, at least 40,000 liters, or at least 50,000 liters, at least 100,000 liters, at least 150,000 liters, at least 200,000 liters, or more. In some embodiments, the agitation container can have a volume of 20,000 liters to 50,000 liters, 20,000 liters to 40,000 liters, 20,000 liters to 30,000 liters, 30,000 liters to 50,000 liters, 30,000 liters to 40,000 liters, or 40,000 liters to 50,000 liters, or more.

[0132] In some embodiments, the stirring vessel may have any combination of the following characteristics. In some embodiments, the stirring vessel may have two impellers. In some embodiments, the impellers are Rushton blade impellers. In some embodiments, the impellers are separated from each other by a distance at least equal to the minimum impeller diameter. In some embodiments, the impellers are 30 inches to 40 inches, 33 inches to 37 inches, 33 inches, 34 inches, 35 inches, 36 inches, or 37 inches from tip to tip. In some embodiments, the stirring vessel has a volume of at least 10,000 liters, at least 20,000 liters, at least 30,000 liters, at least 40,000 liters, or at least 50,000 liters. In some embodiments, the inner diameter of the stirring vessel is 90 inches to 110 inches, 95 inches to 105 inches, 98 inches, 99 inches, 100 inches, 101 inches, or 102 inches. In some embodiments, the first impeller is located 15 inches to 20 inches, 16 inches to 19 inches, or 17 inches to 18 inches from the bottom of the agitation vessel, and the second impeller is located 60 inches to 80 inches, 65 inches to 75 inches, 68 inches, 69 inches, 70 inches, 71 inches, 72 inches, 73 inches, 74 inches, or 75 inches above the first impeller. In some embodiments, the lysed cell composition is agitated at at least 50 rpm, at least 60 rpm, or at least 70 rpm. In some embodiments, the lysed cell composition is agitated at 50 rpm to 70 rpm, 50 rpm to 60 rpm, 60 rpm to 70 rpm, 70 rpm to 100 rpm, 100 rpm to 150 rpm, 150 rpm to 200 rpm, 200 rpm to 250 rpm, or more.

[0133] In some embodiments, by pumping a cell composition, a cracked cell composition or a lipid from a container, a cell composition, a cracked cell composition or a lipid is harvested from the container. In some embodiments, without stirring the container, a cell composition, a cracked cell composition or a lipid is harvested from the container. In some embodiments, by pumping a cell composition, a cracked cell composition or a lipid from a container without stirring, a cell composition, a cracked cell composition or a lipid is harvested from the container. In some embodiments, a cell composition, a cracked cell composition or a lipid is harvested from a container without blowing air. In some embodiments, harvesting the cell composition, lysed cell composition, or lipid by the above techniques produces a crude lipid having a low anisidine value (e.g., 26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 or less) and / or a low phosphorus content (e.g., 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm or less).

[0134] In some embodiments, separating comprises centrifuging the treated cell composition or the treated lysed cell composition (e.g., at a temperature of 30°C to 100°C), thereby separating lipids from the treated cell composition or the treated lysed cell composition by centrifugation.

[0135] In some embodiments, a method comprises centrifuging a treated cell composition or a treated lysed cell composition at a temperature of at least 10°C, at least 20°C, at least 25°C, at least 30°C, at least 35°C, at least 40°C, at least 45°C, at least 50°C, at least 55°C, at least 60°C, at least 65°C, at least 70°C, at least 75°C, at least 80°C, at least 85°C, at least 90°C, at least 95°C, or at least 100°C. In some embodiments, a method comprises centrifuging a treated cell composition or a treated lysed cell composition at a temperature of 10°C to 100°C, 10°C to 90°C, 10°C to 80°C, 20°C to 100°C, 20°C to 90°C, 20°C to 80°C, 25°C to 100°C, 25°C to 90°C, 25°C to 80°C, 25°C to 75°C, 30°C to 100°C, 30°C to 90°C, 30°C to 80°C, 40°C to 100°C, 40°C to 90°C, 40°C to 80°C, 50°C to 100°C, 50°C to 90°C, 50°C to 80°C, 50°C to 70°C, 60°C to 100°C, 60°C to 90°C, 60°C to 80°C, 60°C to 70°C, 70°C to 100°C, or 70°C to 90°C.

[0136] In some embodiments, centrifugation is performed at 1 kilogram per minute (kg / min) to 500 kg / min, 1 kg / min to 400 kg / min, 1 kg / min to 300 kg / min, 1 kg / min to 200 kg / min, 1 kg / min to 100 kg / min, 1 kg / min to 75 kg / min, 1 kg / min to 50 kg / min, 1 kg / min to 40 kg / min, 1 kg / min to 30 kg / min, 1 kg / min to 25 kg / min, 1 kg / min to to 10kg / min, 10kg / min to 500kg / min, 10kg / min to 400kg / min, 10kg / min to 300kg / min, 10kg / min to 200kg / min, 10kg / min to 100kg / min, 10kg / min to 75kg / min, 10kg / min to 50kg / min, 10kg / min to 40kg / min, 10kg / min to 30kg / min, 20kg / min to 500kg / min, 20kg / min to in to 400kg / min, 20kg / min to 300kg / min, 20kg / min to 200kg / min, 20kg / min to 100kg / min, 20kg / min to 75kg / min, 20kg / min to 50kg / min, 20kg / min to 40kg / min, 25kg / min to 500kg / min, 25kg / min to 400kg / min, 25kg / min to 300kg / min, 25kg / min to 200kg / min, 25 The centrifuge is carried out at a feed rate (treated cell composition or treated lysed cell composition entering the centrifuge) of 100 kg / min to 100 kg / min, 25 kg / min to 75 kg / min, 25 kg / min to 50 kg / min, 30 kg / min to 60 kg / min, 30 kg / min to 50 kg / min, 30 kg / min to 40 kg / min, 50 kg / min to 500 kg / min, 100 kg / min to 500 kg / min, or 200 kg / min to 500 kg / min.

[0137] The total time required for separation can vary depending on the volume of treated cell composition or treated lysed cell composition. Typical total time for separation (e.g., centrifugation time) is at least 30 seconds, at least 60 seconds, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 0.1 hour, at least 0.2 hour, at least 0.5 hour, at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 10 hours, at least 12 hours, or 0.1 hour to 24 hours, 0.5 hour to 24 hours, 1 hour to 12 hours, 2 hours to 10 hours, or 4 hours to 8 hours.

[0138] In some embodiments, the method of the present invention comprises 1,000 g to 25,000 g, 1,000 g to 20,000 g, 1,000 g to 10,000 g, 2,000 g to 25,000 g, 2,000 g to 20,000 g, 2,000 g to 15,000 g, 3,000 g to 25,000 g, 3,000 g to 20,000 g, 5,000 g to 25,000 g, 5,000 g to 20,000 g, 5,000 g to 15,000 g, 5 The treated cell composition or the treated lysed cell composition can be centrifuged at a centrifugal force of at least 1,000 g to 10,000 g, 5,000 g to 8,000 g, 10,000 g to 25,000 g, 15,000 g to 25,000 g, or at least 1,000 g, at least 2,000 g, at least 4,000 g, at least 5,000 g, at least 7,000 g, at least 8,000 g, at least 10,000 g, at least 15,000 g, at least 20,000 g, or at least 25,000 g. As used herein, "g" refers to standard gravity or approximately 9.8 m / s 2 In some embodiments, the methods of the invention comprise centrifuging the treated cell composition or the treated lysed cell composition at 4,000 rpm to 14,000 rpm, 4,000 rpm to 10,000 rpm, 6,000 rpm to 14,000 rpm, 6,000 rpm to 12,000 rpm, 8,000 rpm to 14,000 rpm, 8,000 rpm to 12,000 rpm, or 8,000 rpm to 10,000 rpm.

[0139] In some embodiments, the methods of the present invention include drying the lipid after isolating the lipid from the treated cell composition or the treated lysed cell composition to remove water from the lipid. In some embodiments, drying the lipid may include, but is not limited to, heating the lipid to evaporate the water. In some embodiments, after drying, the water content of the lipid is less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, less than 0.1%, or 0% by weight of the lipid. In some embodiments, after drying, the water content of the lipid is between 0% and 3%, 0% and 2.5%, 0% and 2%, 0% and 1.5%, 0% and 1%, 0% and 0.5%, 0.1% and 3%, 0.1% and 2.5%, 0.1% and 2%, 0.1% and 1.5%, 0.1% and 1%, 0.1% and 0.5%, 0.5% and 3%, 0.5% and 2.5%, 0.5% and 2%, 0.5% and 1.5%, 0.5% and 1%, 1% and 3%, 1% and 2.5%, 1% and 2%, 1% and 1.5%, 1.5% and 3%, 1.5% and 2.5%, 1.5% and 2%, 2% and 3%, 2% and 2.5%, or 2.5% and 3%.

[0140] In some embodiments, the method also includes refining lipids by one or more processes selected from caustic refining, degumming, alkali refining, bleaching, deodorization, deacidification, etc., and their combination, to remove one or more phospholipids, free fatty acids, phospholipids, chromatosomes, sterols, odors and other impurities. As used herein, "refined oily matter" is a crude lipid or crude oily matter that has been refined. In some embodiments, the method of the present invention improves the yield of RBD (refined, bleached, deodorized) or RBDW (refined, bleached, deodorized and winterized) oily matter produced by using the lipid extracted by the present invention. In various embodiments, yield is improved in one or more steps of the refining, bleaching, deodorization or winterization steps in refining.

[0141] As used herein, "crude lipid" or "crude oil" is a lipid or oil that has not been refined. In some embodiments, the lipid isolated from the disrupted cell composition is a crude lipid.

[0142] Figure 1 Various exemplary methods of the present invention are schematically described.

[0143] In some embodiments, the method of the present invention comprises concentrating the culture fluid containing microbial cells and / or concentrating the cell composition of lysis. As used herein, "concentrating" refers to removing water from the composition. Concentrating may include, but is not limited to, evaporation, chemical drying, centrifugation, etc., and combinations thereof.

[0144] In some embodiments, the culture fluid comprising microbial cells is concentrated to provide a lipid concentration of at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30% by weight of the culture fluid. In some embodiments, the culture fluid comprising microbial cells is concentrated to provide a lipid concentration of 4% to 40%, 4% to 30%, 4% to 20%, 4% to 15%, 5% to 40%, 5% to 30%, 5% to 20%, 10% to 40%, 10% to 30%, 10% to 20%, 15% to 40%, 15% to 30%, 20% to 40%, 20% to 30%, 25% to 40%, or 30% to 40% by weight of the culture fluid.

[0145] In some embodiments, the cell composition or lysed cell composition is concentrated to provide a lipid concentration of at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 55% by weight of the lysed cell composition. In some embodiments, the cell composition or lysed cell composition is concentrated to provide a lipid concentration of 4% to 40%, 4% to 30%, 4% to 20%, 4% to 15%, 5% to 40%, 5% to 30%, 5% to 20%, 10% to 40%, 10% to 30%, 10% to 20%, 15% to 40%, 15% to 30%, 20% to 40%, 20% to 30%, 25% to 40%, or 30% to 40% by weight of the lysed cell composition.

[0146] In some embodiments, the lipids prepared by the methods of the present invention have an overall aroma intensity of 2 or less. As used herein, the term "overall aroma intensity" refers to the olfactory sensory rating given to the lipids by a panel of sensory analysts. As used herein, the term "sensory analyst" refers to a trained individual who provides feedback and / or ratings on the sensory characteristics of a substance.

[0147] In some embodiments, lipids prepared by the methods of the present invention have an overall aroma intensity of 3 or less. As used herein, the term "overall aroma intensity" refers to the taste or flavor sensory rating given to lipids by a group of sensory analysts. In some embodiments, a general spectral descriptive analysis method is used to assess the aroma and aroma characteristics of a sample. The method uses an intensity scale of 0-15, where 0 = not detected and 15 = very high intensity, to measure the aroma and aroma attributes of an oil.

[0148] In some embodiments, the lipids prepared by the methods of the present invention do not have an aftertaste characterized by a fishy taste.As used herein, the term "aftertaste" refers to the persistence of flavor perception in lipids as characterized by a panel of sensory analysts.

[0149] In some embodiments, the methods of the present invention provide a crude lipid having a peroxide value (PV) of 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less. As used herein, the term "peroxide value" or "PV" refers to a measure of the primary reaction products (e.g., peroxides and hydroperoxides) that occur during lipid oxidation. In some embodiments, PV is an indicator of lipid quality, and the degree of oxidation that occurs in lipids with a low PV (i.e., 5 or less) shows increased stability and sensory characteristics over lipids with a PV greater than 5. In some embodiments, adding a base to the lysed cell composition as described above increases the pH of the lysed cell composition and inhibits lipid oxidation, thereby minimizing the number of free radicals in the lysed cell composition, so that the crude lipid obtained from the method of the present invention has a low PV (i.e., 5 or less).

[0150] In some embodiments, the methods of the present invention provide a crude lipid having an anisidine value (AV) of 26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 or less. As used herein, the term "anisidine value" or "AV" refers to a measure of secondary reaction products (e.g., aldehydes and ketones) that occur during lipid oxidation. In some embodiments, AV is an indicator of lipid quality and the degree of oxidation that has occurred in the lipid. Lipids with a low AV (i.e., 26 or less) exhibit increased stability and organoleptic characteristics compared to lipids with an AV greater than 26. In some embodiments, adding a base to the lysed cell composition as described above increases the pH of the lysed cell composition and inhibits lipid oxidation, thereby minimizing the number of free radicals in the lysed cell composition, so that the crude lipid obtained from the method of the present invention has a low AV (i.e., 26 or less).

[0151] In some embodiments, the methods of the invention provide a crude lipid having a phosphorus content of 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less, 80 ppm or less, 75 ppm or less, 70 ppm or less, 65 ppm or less, 60 ppm or less, 55 ppm or less, 50 ppm or less, 45 ppm or less, 40 ppm or less, 35 ppm or less, 30 ppm or less, 25 ppm or less, 20 ppm or less, 15 ppm or less, 10 ppm or less, 5 ppm or less, 4 ppm or less, 3 ppm or less, 2 ppm or less, or 1 ppm or less.

[0152] In some embodiments, the methods of the invention provide for extraction more efficiently than if the extraction were performed using a solvent (e.g., a typical hexane extraction or process (Westfalia Separator AG, Germany)) to produce crude lipids with lower anisidine values, lower peroxide values, lower phosphorus contents and / or higher extraction yields. The process is one of extracting lipids with a water-miscible organic solvent as described in US Pat. No. 5,928,696 and International Publication Nos. WO 01 / 76385 and WO 01 / 76715, each of which is incorporated herein by reference in its entirety.

[0153] In some embodiments, heating the lysed cell composition causes secondary reaction products (e.g., aldehydes and ketones) to participate in a reaction similar to the Maillard reaction with proteins present in the lysed cell composition. The reaction is believed to produce products with antioxidant activity, which reduces the oxidation of lipids. In some embodiments, additional proteins, such as soy protein, can be added to the lysed cell composition to increase antioxidant activity. The reduction in lipid oxidation reduces the AV of lipids, reduces any aftertaste of lipids and / or increases the stability of lipids. In some embodiments, stability is increased by at least 5%, at least 10%, at least 15%, or at least 20%.

[0154] In some embodiments, lipids extracted by the methods of the present invention, biomass remaining after lipid extraction, or a combination thereof can be used directly as food or food ingredients, such as baby food, infant formula, beverages, sauces, dairy-based foods (e.g., milk, yogurt, cheese, and ice cream), oils (e.g., cooking oils or salad dressings), and baked goods; nutritional supplements (e.g., in capsule or tablet form); feed or feed supplements for any non-human animal (e.g., an animal whose products (e.g., meat, milk, or eggs) are consumed by humans); food supplements; and drugs (in direct or adjunctive therapeutic applications); and ingredients in biofuels. The term "animal" refers to any organism belonging to the animal kingdom, and includes any human animal, as well as non-human animals from which products (e.g., milk, eggs, poultry, beef, pork, or lamb) are derived. In some embodiments, lipids and / or biomass can be used in seafood. Seafood is derived from, but not limited to, fish, shrimp, and shellfish. The term "product" includes any product derived from such an animal, including, but not limited to, meat, eggs, milk, or other products. When lipids and / or biomass are fed to such animals, polyunsaturated lipids may be incorporated into the meat, milk, eggs or other products of such animals to increase their content of these lipids.

[0155] Lipid composition

[0156] In some embodiments, the invention relates to microbial lipids extracted according to the methods of the invention. In some embodiments, the microbial lipids have an anisidine value of 26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 or less, and / or a peroxide value of 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less and / or a peroxide value of 100 ppm or less, 95 ppm or less, 90 ppm or less, 85 ppm or less. In some embodiments, the lipid has a phosphorus content of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 50%, at least 60%, at least 65 ... In some embodiments, the lipids have at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% DHA by weight, and / or at least 10%, at least 15%, or at least 20% DPA n-6 by weight, and / or at least 10%, at least 15%, or at least 20% EPA by weight, and / or at least 10%, at least 15%, or at least 20% ARA by weight. In some embodiments, lipids extracted according to the methods of the invention result in a higher yield than if the extraction were performed using a solvent (e.g., a typical hexane extraction or process (Westfalia Separator AG, Germany)) lower anisidine value, lower peroxide value, lower phosphorus content and / or higher extraction yield.

[0157] The microbial lipids of the present invention can be any lipid derived from a microorganism, including, for example: a crude oil extracted from the biomass of a microorganism without further processing; a refined oil obtained by treating a crude microbial oil with further processing steps such as refining, bleaching and / or deodorization; a diluted microbial oil obtained by diluting a crude or refined microbial oil; or an enriched oil obtained, for example, by treating a crude or refined microbial oil with a further purification method to increase the concentration of fatty acids (e.g., DHA) in the oil.

[0158] In some embodiments, the microbial lipid comprises a sterol ester fraction of 0%, at least 0.1%, at least 0.2%, at least 0.5%, at least about 1%, at least 1.5%, at least 2%, or at least 5% by weight. In some embodiments, the microbial lipid comprises a sterol ester fraction of 0% to 1.5%, 0% to 2%, 0% to 5%, 1% to 1.5%, 0.2% to 1.5%, 0.2% to 2%, or 0.2% to 5%. In some embodiments, the microbial lipid comprises a sterol ester fraction of less than 5%, less than 4%, less than 3%, or less than 2% by weight.

[0159] In some embodiments, the microbial lipid comprises at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% triglyceride fraction by weight. In some embodiments, the microbial lipid comprises 65% to 95%, 75% to 95%, or 80% to 95%, or 97% by weight, or 98% by weight triglyceride fraction.

[0160] In some embodiments, the microbial lipids comprise at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 2.5%, or at least 5% free fatty acid (FFA) fraction by weight. In some embodiments, the microbial lipids comprise 0.5% to 5%, 0.5% to 2.5%, 0.5% to 2%, 0.5% to 1.5%, 0.5% to 1%, 1% to 2.5%, 1% to 5%, 1.5% to 2.5%, 2% to 2.5%, or 2% to 5% free fatty acid fraction by weight. In some embodiments, the microbial lipids comprise less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% free fatty acid fraction by weight.

[0161] In some embodiments, the microbial lipid comprises at least 0.5%, at least 1%, at least 1.5%, at least 2%, or at least 5% sterol fraction by weight. In some embodiments, the microbial lipid comprises 0.5% to 1.5%, 1% to 1.5%, 0.5% to 2%, 0.5% to 5%, 1% to 2%, or 1% to 5% sterol fraction by weight. In some embodiments, the microbial lipid comprises less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% sterol fraction by weight.

[0162] In some embodiments, the microbial lipid comprises at least 1.5%, at least 2%, at least 2.5%, at least 3%, at least 3.5%, or at least 5% by weight of a diacylglycerol fraction. In some embodiments, the microbial lipid comprises between 1.5% and 3%, between 2% and 3%, between 1.5% and 3.5%, between 1.5% and 5%, between 2.5% and 3%, between 2.5% and 3.5%, or between 2.5% and 5% by weight of a diacylglycerol fraction.

[0163] In some embodiments, the microbial lipid comprises less than 2%, less than 1.5%, less than 1%, or less than 0.5% unsaponifiable matter by weight of the oil.

[0164] Lipids present in the microbial oil, such as the triglyceride fraction, can be separated by flash chromatography and analyzed by thin layer chromatography (TLC), or separated and analyzed by other methods known in the art.

[0165] In some embodiments, the microbial lipids and / or one or more fractions thereof selected from the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, and combinations thereof comprise at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% DHA by weight. In some embodiments, the microbial lipids and / or one or more fractions thereof selected from the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, and combinations thereof comprise 40% to 45%, 40% to 50%, 40% to 60%, 50% to 60%, 55% to 60%, 40% to 65%, 50% to 65%, 55% to 65%, 40% to 70%, 40% to 80%, 50% to 80%, 55% to 80%, 60% to 80%, or 70% to 80% DHA by weight. In some embodiments, the microbial lipids comprise a sterol ester fraction comprising 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 13% or less DHA by weight. In some embodiments, the microbial lipids and / or one or more fractions thereof selected from the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, and combinations thereof comprise 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less EPA by weight. In some embodiments, the microbial lipids and / or one or more fractions thereof selected from the triglyceride fraction, the free fatty acid fraction, the sterol fraction, the diglyceride fraction, and combinations thereof comprise 2% to 3%, 2% to 3.5%, 2.5% to 3.5%, 2% to 6%, 2.5% to 6%, 3.0% to 6%, 3.5% to 6%, 5% to 6%, or 2% to 10% EPA by weight. In some embodiments, the microbial lipids and / or one or more fractions thereof selected from sterol ester fractions, triglyceride fractions, free fatty acid fractions, sterol fractions, diglyceride fractions, polar fractions (including phospholipid fractions), and combinations thereof are substantially free of EPA. In some embodiments, the microbial lipids and / or one or more fractions thereof selected from sterol ester fractions, triglyceride fractions, free fatty acid fractions, sterol fractions, diglyceride fractions, polar fractions (including phospholipid fractions), and combinations thereof include a weight ratio of DHA to EPA of at least 5:1, at least 7:1, at least 9:1, at least 10:1, at least 15:1, at least 20:1, at least 25:1, at least 30:1, or at least 50:1, wherein the microbial lipids and / or one or more fractions thereof comprise 10% or less EPA by weight.In some embodiments, the microbial lipids and / or one or more fractions thereof selected from the group consisting of sterol ester fractions, triglyceride fractions, free fatty acid fractions, sterol fractions, diglyceride fractions, polar fractions (including phospholipid fractions), and combinations thereof comprise a weight ratio of DHA to EPA of at least 5:1 but less than 20:1. In some embodiments, the weight ratio of DHA to EPA is 5:1 to 18:1, 7:1 to 16:1, or 10:1 to 15:1. In some embodiments, the microbial lipids and / or one or more fractions thereof selected from the group consisting of sterol ester fractions, triglyceride fractions, free fatty acid fractions, sterol fractions, diglyceride fractions, polar fractions (including phospholipid fractions), and combinations thereof comprise 0.1% to 0.25%, 0.2% to 0.25%, 0.1% to 0.5%, or 0.1% to 1.5% ARA by weight. In some embodiments, microbial lipid and / or it is selected from sterol ester fraction, triglyceride fraction, free fatty acid fraction, sterol fraction, diglyceride fraction, polar fraction (comprising phospholipid fraction), and one or more fractions in their combination contain 1.5% or less, 1% or less, 0.5% or less, 0.2% or less or 0.1% or less ARA by weight. In some embodiments, microbial lipid and / or it is selected from sterol ester fraction, triglyceride fraction, free fatty acid fraction, sterol fraction, diglyceride fraction, polar fraction (comprising phospholipid fraction), and one or more fractions in their combination are substantially free of ARA. In some embodiments, the microbial lipids and / or one or more fractions thereof selected from a sterol ester fraction, a triglyceride fraction, a free fatty acid fraction, a diglyceride fraction, a polar fraction (including a phospholipid fraction), and combinations thereof comprise a weight ratio of DHA to ARA of at least 20: 1, at least 30: 1, at least 35: 1, at least 40: 1, at least 60: 1, at least 80: 1, at least 100: 1, at least 150: 1, at least 200: 1, at least 250: 1, or at least 300: 1. In some embodiments, the microbial lipids and / or one or more fractions selected from a sterol ester fraction, a triglyceride fraction, a free fatty acid fraction, a sterol fraction, a diglyceride fraction, a polar fraction (including a phospholipid fraction), and combinations thereof comprise 0.5% to 1%, 0.5% to 2%, 0.5% to 2.5%, 0.5% to 3%, 0.5% to 3.5%, 0.5% to 5%, 0.5% to 6%, 1% to 2%, 2% to 3%, 2% to 3.5%, 1% to 2.5%, 1% to 3%, 1% to 3.5%, 1% to 5%, or 1% to 6% DPA n-6 by weight.In some embodiments, the microbial lipids and / or one or more fractions thereof selected from the group consisting of sterol ester fractions, triglyceride fractions, free fatty acid fractions, sterol fractions, diglyceride fractions, polar fractions (including phospholipid fractions), and combinations thereof contain 6% or less, 5% or less, 3% or less, 2.5% or less, 2% or less, 1% or less, or 0.5% or less DPA n-6 by weight. In some embodiments, the microbial lipids and / or one or more fractions thereof selected from the group consisting of sterol ester fractions, triglyceride fractions, free fatty acid fractions, sterol fractions, diglyceride fractions, polar fractions (including phospholipid fractions), and combinations thereof are substantially free of DPA n-6. In some embodiments, the microbial lipids and / or one or more fractions thereof selected from a sterol ester fraction, a triglyceride fraction, a free fatty acid fraction, a sterol fraction, a diglyceride fraction, a polar fraction (including a phospholipid fraction), and combinations thereof comprise a weight ratio of DHA to DPA n-6 of greater than 6: 1, or at least 8: 1, at least 10: 1, at least 15: 1, at least 20: 1, at least 25: 1, at least 50: 1, or at least 100: 1. In some embodiments, the microbial lipids and / or one or more fractions thereof selected from a sterol ester fraction, a triglyceride fraction, a free fatty acid fraction, a sterol fraction, a diglyceride fraction, a polar fraction (including a phospholipid fraction), and combinations thereof contain 5% or less, 4% or less, 3% or less, 2% or less, 1.5% or less, 1% or less, or 0.5% or less by weight of linoleic acid (18:2n-6), linolenic acid (18:3n-3), eicosenoic acid (20:1n-9), and erucic acid (22:1n-9). In some embodiments, the microbial lipids and / or one or more fractions thereof selected from the group consisting of sterol ester fractions, triglyceride fractions, free fatty acid fractions, sterol fractions, diglyceride fractions, polar fractions (including phospholipid fractions), and combinations thereof comprise 5% or less, 4% or less, 3% or less, 2% or less, 1.5% or less, or 1% or less heptadecanoic acid (17:0) by weight. In some embodiments, the microbial lipids and / or one or more fractions thereof comprise 0.01% to 5%, 0.05% to 3%, or 0.1% to 1% heptadecanoic acid by weight.

[0166] In some embodiments, the extracted microbial lipid comprises at least 70% by weight of a triglyceride fraction, wherein the docosahexaenoic acid content of the triglyceride fraction is at least 50% by weight, wherein the docosapentaenoic acid n-6 ​​content of the triglyceride fraction is at least 0.5% by weight to 6% by weight, and wherein the anisidine value of the oil is 26 or less. In some embodiments, the extracted microbial lipid comprises at least 70% by weight of a triglyceride fraction, wherein the docosahexaenoic acid content of the triglyceride fraction is at least 40% by weight, wherein the docosapentaenoic acid n-6 ​​content of the triglyceride fraction is at least 0.5% by weight to 6% by weight, wherein the ratio of docosahexaenoic acid to docosapentaenoic acid n-6 ​​is greater than 6:1, and wherein the lipid has an anisidine value of 26 or less. In some embodiments, the extracted microbial lipid comprises a triglyceride fraction of at least 70% by weight, wherein the docosahexaenoic acid content of the triglyceride fraction is at least 60% by weight, and wherein the lipid has an anisidine value of 26 or less. In some embodiments, the extracted microbial lipid having any of the above fatty acid profiles has an anisidine value of 26 or less, 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 2 or less, or 1 or less, and / or a peroxide value of 5 or less, 4.5 or less, 4 or less, 3.5 or less, 3 or less, 2.5 or less, 2 or less, 1.5 or less, 1 or less, 0.5 or less, 0.2 or less, or 0.1 or less, and / or a peroxide value of 100 ppm or less, 95 ... In some embodiments, the extracted microbial lipids having any of the above fatty acid profiles are extracted from a Thraustochytrium microorganism having the characteristics of a Thraustochytrium species deposited with ATCC Accession No. PTA-9695, PTA-9696, PTA-9697, or PTA-9698. In some embodiments, the extracted microbial lipids having any of the above fatty acid profiles are extracted from a Thraustochytrium microorganism having the characteristics of a Thraustochytrium species deposited with ATCC Accession No. PTA-9695, PTA-9696, PTA-9697, or PTA-9698. In some embodiments, the extracted microbial lipids having any of the above fatty acid profiles are crude lipids. In some embodiments, the crude lipids have less than 5% organic solvent by weight or volume. In some embodiments, the microbial lipids extracted according to the methods of the invention result in a higher yield than if the extraction were performed using a solvent (e.g., a typical hexane extraction or process (Westfalia Separator AG, Germany)) lower anisidine value, lower peroxide value, lower phosphorus content and / or higher extraction yield.

[0167] Having generally described the present invention, further understanding can be obtained by reference to the examples provided herein. These examples are provided for illustrative purposes only and are not intended to be restrictive. The following examples are non-restrictive illustrations of the method of the present invention and of the lipids prepared by the method of the present invention. Other suitable modifications and adjustments to the various conditions and parameters commonly encountered when extracting lipids from cells will be apparent to those skilled in the art and are within the spirit and scope of the present invention.

[0168] Experimental example

[0169] Example 1. Pilot test (AEX-O-1150-tank B)

[0170] 524.6 kg of fermentation broth containing about 10% w / w Schizochytrium microbial cells was heated to 60°C and the pH was adjusted to 8.3 by adding 50% NaOH (available from Colonial Chemicals Solutions, 916 W Lathrop Ave, Savannah, GA 31415). 2.4FG (available from Novozymes, Franklinton, NC) was added to the culture broth for enzymatic lysis, and after 30 minutes, the pH drifted to 6.9 and was readjusted to 8.1. After 2 hours of enzymatic lysis, the demulsification process was started by raising the pH to 10.5 and raising the temperature to 90°C. When the temperature reached 90°C, the pH drifted to 8.0 and was readjusted to 10.2. After 2 hours of demulsification, 10.49 kg of NaCl salt was added. 30 minutes after the salt was added, the pH was reduced to 5.4 by adding 50% citric acid (prepared in house from solid citric acid (available from Tate & Lyle, 2200E Eldorado St, Decatur Illinois USA, 62521)). After 30 minutes, the pH increased to 9.7. After 1.5 hours, 15 ml of the composition was centrifuged at about 11000xg for 3 minutes, and it was found that the demulsification was complete. The temperature was reduced to 77°C and the pH was reduced to 8.3 for centrifugation. The demulsified culture broth was centrifuged at about 8000 x g on a three-phase pilot centrifuge. 29.6 kg of a light phase consisting mainly of crude oil, 538.6 kg of a heavy phase consisting mainly of fermentation medium and some residual cells, and 2.2 kg of a third phase consisting mainly of fermentation medium and some cell debris were collected.

[0171] Example 2. Pilot test (AEX-O-1150-tank C)

[0172] 525.2 kg of fermentation broth containing about 10% w / w Schizochytrium microbial cells (the same batch of fermentation broth as in tank B in Example 1) was heated to 60°C and the pH was adjusted to 7.9 by adding 50% NaOH (available from Colonial Chemicals Solutions, 916 W Lathrop Ave, Savannah, GA 31415). 2.4FG (available from Novozymes, Franklinton, NC) was added to the culture broth for enzymatic lysis. After 30 minutes, the pH drifted to 6.7 and was readjusted to 8.1. After 2 hours of enzymatic lysis, the demulsification process was started by raising the pH to 10.5 and raising the temperature to 90°C. When the temperature reached 90°C, the pH was readjusted to 10.3. After 1.5 hours of demulsification, 10.5 kg of NaCl salt was added. 30 minutes after the salt was added, the temperature was reduced to 85°C and the pH was reduced to 5.5 by adding 75% phosphoric acid (available from Tate & Lyle, 2200E Eldorado St, Decatur Illinois USA, 62521). After 30 minutes, the pH increased to 9.9. After 2 hours, the pH was reduced to 6.4. After 1.5 hours, the pH was raised to 8.1, and 15 ml of the composition was centrifuged to check the demulsification level. Demulsification was found to be incomplete and the contents of the tank were left at 80°C overnight.

[0173] On the second day, the temperature was raised to 85°C and the pH was lowered to 6.4. After 30 minutes, the pH was raised to 7.8 and about 90% demulsification was achieved. The demulsified culture broth was centrifuged at about 8000 x g on a three-phase pilot centrifuge. 31.6 kg of a light phase (mainly composed of crude oil) containing 2.46% water, 550.6 kg of a heavy phase (mainly composed of fermentation medium and some residual cells) containing 82.1% water, and 16.4 kg of a third phase (mainly composed of fermentation medium and some cell debris) containing 75.7% water were collected.

[0174] Table 1. Free fatty acids, peroxide values ​​and anisidine values ​​of Example 1 and Example 2

[0175] Experimental run Free fatty acids (%) Peroxide value (meq / kg) Anisidine value AEX-O-1150 Slot B 2.68 0.39 10.6 AEX-O-1150 Slot C 1.97 0.46 8.2

[0176] Table 2. Data from laboratory RBWD (refined, bleached, winterized, deodorized) of Experimental Examples 1 and 2

[0177] batch number AEX-1150 Slot C AEX-1150 Slot B Crude oil Free fatty acids (%) 1.97 2.68 Peroxide value (meq / kg) 0.46 0.39 Anisidine value 10.60 6.20 Refined oil Yield 92.1％ 94.5％ soap 121.8 91.3 Free fatty acids (%) 0.06 0.08 Peroxide value (meq / kg) 0.67 0.73 Anisidine value 12.40 10.00 Bleached oil Yield 94.5％ 93.8％ soap 0.0 0.0 Free fatty acids (%) 0.42 0.56 Peroxide value (meq / kg) 1.21 0.81 Anisidine value 11.80 9.90 color 2.8R, 18.0Y 0.9R, 7.4Y Winterized oil Yield 90.0％ 88.7％

[0178] Example 3. Pilot test

[0179] 1797.6 kg of fermentation broth containing about 10% w / w Schizochytrium microbial cells was heated to 60°C and the pH was adjusted to 8.0 by adding 50% NaOH (available from Colonial Chemicals Solutions, 916 W Lathrop Ave, Savannah, GA 31415). 2.4FG (available from Novozymes, Franklinton, NC) was added to the culture broth for enzymatic lysis for two hours. The pH of the lysed composition was adjusted to 10.5 and heated to 90°C. When the temperature reached 90°C, the pH drifted to 8.8 and the pH was adjusted to 10.1. After 1 hour at 90°C, the temperature was reduced to 82°C and the pH was reduced to 5.2 for 30 minutes by adding 50% citric acid (prepared in-house from solid citric acid (available from Tate & Lyle, 2200E Eldorado St, Decatur Illinois USA, 62521)). The pH was then raised to 10.1 for 40 minutes and the pH was again reduced to 5.4. After 30 minutes, the pH was raised to 8.2. The 15 ml sample was centrifuged at about 11000xg for 3 minutes, and the demulsification was satisfactory. The demulsified culture broth was centrifuged at about 8000xg on a three-phase pilot centrifuge. 78.9 kg of a light phase consisting mainly of crude oil, 1876.6 kg of a heavy phase consisting mainly of fermentation medium and some residual cells, and 10.6 kg of a third phase consisting mainly of fermentation medium and some cell debris were collected. The crude oil was dried to 0.27% moisture and processed in RBWD.

[0180] Table 3. Data from pilot RBWD (refined, bleached, winterized, deodorized) of Example 3

[0181] RBD-O-1176 Refined oil Starting weight (kg) 70.7 water(%) 5 Soap (ppm) 334.84 Free fatty acids (%) 0.28 Peroxide value (meq / kg) 0.4 Yield (%) 94.3 Bleached oil Starting weight (kg) 66.68 Yield (%) 90.19 Winterized oil Starting weight (kg) 60.14 Free fatty acids (%) 0.16 Peroxide value (meq / kg) 0.25 Yield (%) 87.4 Deodorized oil Starting weight (kg) 52.6 Final weight (kg) 52.1 Free fatty acids (%) 0.08 Peroxide value (meq / kg) 0.27 Yield (%) 99.00

Claims

1. A method for obtaining lipids from a composition comprising microbial cells, the method comprising: a) heating the composition comprising microbial cells to a temperature of 60°C to 80°C; b) adding one or more enzymes capable of destroying the cell walls of the microbial cells to the composition obtained in step a) for a time sufficient to lyse the microbial cells to obtain a lysed cell composition; c) heating the lysed cell composition to a temperature of 80° C. to 90° C.; d) adjusting the pH of the composition obtained in step c) to a pH of 10 to 12 by adding a base and maintaining the pH of 10 to 12 for 5 minutes to 2 hours; e) adjusting the pH of the composition obtained in step (d) to a pH of 4 to 6 by adding an acid and maintaining the pH of 4 to 6 for 5 minutes to 2 hours; f) optionally repeating steps (d) and (e) until the composition is sufficiently demulsified; g) separating the lipid from the demulsified lysed cell composition; and h) recovering the lipid; wherein (d) and (e) are repeated 1, 2, 3 or 4 times.

2. The method of claim 1, wherein (a) comprises heating the composition to 80°C.

3. The method of claim 1, wherein (a) and / or (b) further comprises adjusting the pH from 7 to 9.

4. The method of claim 1, wherein the composition is subjected to one or more temperature shock cycles before (d), after (d), before (e), after (e), or a combination thereof.

5. The method of claim 4, wherein the temperature shock comprises adjusting the temperature from 80-90°C to 4-20°C and back to 80-90°C.

6. The method of claim 1, wherein (b), (d) and / or (e) further comprises adding a salt in an amount of 0.05% to 20% by weight of the lysed cell composition.

7. The method of claim 1, wherein (a) further comprises agitating the cells.

8. The method of claim 1, wherein the cells of (a) are unwashed.

9. The method of claim 1, wherein the cells of (a) are contained in a fermentation broth.

10. The method of claim 1, wherein (g) comprises centrifuging the demulsified lysed cell composition.

11. The method of claim 1, wherein the lipid comprises a polyunsaturated fatty acid.

12. The method of claim 11, wherein the polyunsaturated fatty acid is selected from the group consisting of omega-3 fatty acids, omega-6 fatty acids, and mixtures thereof.

13. The method according to claim 11, wherein the polyunsaturated fatty acid is selected from the group consisting of docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), arachidonic acid (ARA), gamma-linolenic acid (GLA), dihomo-gamma-linolenic acid (DGLA), stearidonic acid (SDA), and mixtures thereof.

14. The method of claim 13, wherein the polyunsaturated fatty acid is docosahexaenoic acid (DHA).

15. The method of claim 13, wherein the polyunsaturated fatty acid is eicosapentaenoic acid (EPA).

16. The method of claim 13, wherein the polyunsaturated fatty acid is arachidonic acid (ARA).

17. The method of claim 1, wherein the microbial cells are algal cells.

18. The method of claim 1, wherein the microbial cell is a yeast cell.

19. The method of claim 1, wherein the microbial cell is one or more of a fungal cell, a protist cell, or a bacterial cell.

20. The method of claim 1, wherein the microbial cell is from the genus Mortierella, Crypthecodinium, or Thraustochytridales.

21. The method of claim 20, wherein the microbial cell is from the Thraustochytriales.

22. The method of claim 21, wherein the microbial cell is from the genus Thraustochytrium, Schizochytrium, or a mixture thereof.

23. The method of claim 20, wherein the microbial cell is from Mortierella alpina.

24. The method of claim 1, wherein the lysed cell composition comprises liquid, cell debris, and microbial oil.

25. The method of claim 1, wherein the oil is obtained from the cells without using an organic solvent.

26. The method of claim 1, wherein the enzyme is selected from the group consisting of beta glucanase, xylanase, cellulase, protease, pectinase, mannanase, amylase, and combinations thereof.

27. The method of claim 26, wherein the enzyme is a protease.

28. The method of claim 1, wherein the enzyme is added in an amount of 0.05% to 10% by weight of the lysed cell composition.

29. The method of claim 6, wherein the salt is selected from the group consisting of alkali metal salts, alkaline earth metal salts, sulfate salts, and combinations thereof.

30. The method of claim 1, wherein the lipid of (h) is a crude lipid.

31. The method of claim 30, wherein (h) further comprises refining the crude lipid to obtain refined lipid.

32. The method of claim 1, wherein the lipid comprises at least 30% by weight docosahexaenoic acid.

33. The method of claim 1, wherein the lipid comprises at least 30% eicosapentaenoic acid by weight.

34. The method of claim 1, wherein the lipid comprises at least 30% arachidonic acid by weight.

35. The method of claim 1, wherein the lipid has an anisidine value of less than 26.

36. The method of claim 1, wherein the phosphorus content of the lipid is 100 ppm or less.

37. The method of claim 1, wherein the lipid has a peroxide value of less than 5 meq / kg.

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